Calcium triggers reversal of calmodulin on nested anti-parallel sites inthe IQmotif of the neuronal voltage-dependent sodium channel NaV1.2☆

Liam Hovey a,1, C. Andrew Fowler b,1, Ryan Mahling a, Zesen Lin a, Mark Stephen Miller a, Dagan C. Marx a,Jesse B. Yoder a, Elaine H. Kim a, Kristin M. Tefft a, Brett C. Waite a, Michael D. Feldkamp a,Liping Yu b, Madeline A. Shea a,⁎a Department of Biochemistry, University of Iowa, 52242-1109 Iowa City, United Statesb NMR Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 52242-1109 Iowa City, United States

a b s t r a c ta r t i c l e i n f o

Article history:Received 8 January 2017Received in revised form 23 February 2017Accepted 23 February 2017Available online 9 March 2017

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic cal-cium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted)and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif[IQRAYRRYLLK] binding to apo CaM (~3 nM) and (Ca2+)4-CaM (~85 nM), showing that apo CaM binding is fa-vored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2IQp)exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers confor-mational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMC

bound to NaV1.2IQp. The polarity of (Ca2+)2-CaMC relative to the IQ motif was opposite to that seen in apoCaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal ofCaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation ofCaMN allowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicin-ity of the IQ motif.

© 2017 Elsevier B.V. All rights reserved.

Keywords:Molecular recognitionAllosteryNMRTitrationBindingLinkageBiosensorThermodynamicsFree energySodium channelsFRET

1. Introduction

The human voltage-dependent sodium channel family (NaV)is responsible for the generation and propagation of action potentials[1,2]. NaV channelopathies include epilepsy [3], “Long QT” syndrome[4], familial autism [5] and pain insensitivity [6]. NaV channels arebelieved to undergo calcium-dependent regulation [7] mediated by cal-modulin (CaM), an essential intracellular calcium sensor [8]. However,the mechanisms of calcium-induced regulation remain poorly under-stood, and may vary among NaV subtypes [9].

1.1. Universal design for Apo CaM binding to NaV IQ motifs

The architecture of the Nav1.2 pore-formingα-subunit (2005 a.a.) isshown in Fig. 1A. It has 4 multi-helical transmembrane domains(green cylinders) that form the ion-conducting pore and containvoltage-sensing domains; they are connected by intracellular linkers[10]. The Nav1.2 C-terminal domain (CTD) contains a 4-helix bundle(EF-like or EFL domain) that preferentially binds FGF12 [11] but alsobinds other members of the FGF homology factor (FHF) family. A

Biophysical Chemistry 224 (2017) 1–19

Abbreviations: BAA, basic, amphipathic alpha helix; CaM, calmodulin; CaMBD,calmodulin-binding domain; CaMC, C-domain of CaM; CaMN, N-domain of CaM; CFP,cyan fluorescent protein; CTD, C-terminal domain; EGTA, ethylene glycolbis(aminoethylether)-N,N,N′,N′-tetra-acetic acid; FRET, Förster/fluorescence resonanceenergy transfer; HEPES, N-(2-hydroxy-ethyl)piperazine-N′-2-ethanesulfonic acid; HSQC,heteronuclear single quantum coherence; Nav1.2IQp, IQ motif peptide (residues 1901 to1927) of rat NaV1.2; NaV, voltage-gated sodium channel; NMR, nuclear magneticresonance; NOE, nuclear overhauser effect; NTA, nitrilo-triacetic acid; RMSD, root-mean-squared deviation; YFP, yellow fluorescent protein.☆ These studies were supported by Iowa Center for Research by Undergraduates

Fellowships to L.H., Z.L., and D.C.M., a graduate fellowship for M.S.M. from the Universityof Iowa Center for Biocatalysis and Bioprocessing Training Grant in BiotechnologyNational Institutes of Health (T32 GM08365), a Biochemistry Summer UndergraduateFellowship for B.C.W., the Roy J. Carver Charitable Trust Grant 01-224, and a grant toM.A.S. from the National Institutes of Health (R01 GM57001). The content is solely the re-sponsibility of the authors and does not necessarily represent the official views of theNational Institutes of Health.⁎ Corresponding author.

E-mail address: madeline-shea@uiowa.edu (M.A. Shea).1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.bpc.2017.02.0060301-4622/© 2017 Elsevier B.V. All rights reserved.

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Fig. 1. Schematics and structures of CaM binding to NaV IQ motifs. In all structures of CaM, residues 1–75 are blue, 76–80 are black, and 81–148 are red. A: Cartoon of a voltage-gated sodium channel (NaV) α-subunit (green) with 4 multi-helicaltransmembrane units DI, DII, DIII, and DIV (cylinders). The C-terminal domain (CTD) contains a 4-helix bundle domain dubbed EF-like (EFL) and an IQ motif bound to apo CaMC (red) with apo CaMN (blue) connected by a flexible linker (black).The NaV inactivation gate (purple) links DIII and DIV, and contains a site for binding (Ca2+)4-CaM. B: Solution structures of the NaV1.2 EF-like domain (PDB: 2KAV) depicting residues E1777 (blue sphere) to R1881 (red sphere); cylindricalhelices are colored in shades of green correspond to the sections labeled H in the linear diagram of the NaV1.2 CTD residues 1777 to 1937 below. C: Crystallographic structure of apo (calcium-depleted) CaM bound to a CTD fragment of NaV1.5(PDB: 4DCK); shades of green match panel B. The NaV1.2 CTD in 4DCK was aligned (using PyMOL (Schrödinger, LLC)) to the corresponding residues in 2KAV in panel B based on EFL-located helical residues 1791–1866 and 1855–1866. D:Overlay of structures having apo or Mg2+-bound CaM associated with NaV IQ motifs (PDB: 2KXW, 2L53, 4DCK, 4OVN, and 3WFN). For structures determined by NMR (2KXW and 2L53), only the minimized average structure is shown.Structures aligned with PyMOL (Schrödinger, LLC) based on position of CaM residues 102–112 and 117–128 (helices F and G). E: Structures of individual CaM-IQ complexes in overlay in panel D; Mg2+ ions are gray spheres. For 2L53, alldeposited models consistent with the NMR data are illustrated. Structures were aligned as in panel D. Spheres highlight IQ motif residues at positions 1, 2, 5 and 8. In, Nav1.2IQp these are I1912 (cyan), Q1913 (green), Y1916 and Y1919 (gold). InNav1.5IQp and Nav1.6IQp, colors for corresponding residues match WebLogos in panel F. F: WebLogo 3.3 [26] analyses comparing IQ motif sequences for NaV1.2 from 53 species, NaV1.5 from 40 species and NaV1.6. from 45 species (sequencealignments shown in Supplemental Tables S1A, S1B, S1C). The height of each letter denotes relative conservation of residues. A star indicates the fully conserved Q residue of all three channels which is also labeled in panel E. Within the set ofresidues binding apo CaMC, complete conservation is observed for positions corresponding toQ1913, R1914, A1915, R1917 inNaV1.2. (For interpretation of the references to color in thisfigure legend, the reader is referred to theweb version of this article.)

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solution structure (2KAV [12]) of a fragment of Nav1.2 that includes EFL(Fig. 1B) shows that the next (fifth) helix is connected to EFL by a flex-ible linker, allowing it to adopt many orientations relative to the 4-helixbundle. A schematic diagram of a fragment of the Nav1.2 CTD sequencebelow 2KAV shows the relative length and position of the heliceswithin 2KAV that precede the helix containing the IQ motif(IQxxx[R,K]Gxxx[R,K] [13]) that binds CaM tightly [14–16]. The IQmotif is necessary for several members of the human NaV family tomaintain a closed, inactivated state [17,18].

Five high-resolution structures of apo (calcium-depleted) CaMbound to NaV IQ motifs are available. In all of these structures, CaMC

adopts the semi-open tertiary structure also observed in complexes ofapo CaMC bound to myosin V [19] and the SK Channel [20]. A crystalstructure of apo CaM bound to a CTD fragment of cardiac NaV1.5(4DCK, Fig. 1C) shows the C-domain of CaM (CaMC) binds to the IQmotif, while the N-domain (CaMN) makes few contacts with any partof the CTD. An overlay of this complex with apo CaM bound to the IQmotifs from NaV1.2 [16], NaV1.5 [21–23] and NaV1.6 [24] (Fig. 1D) illus-trates that the position of apo CaMN is highly variable relative to CaMC.

The five structures are shown individually in Fig. 1E (note that CaMN

is not shown for Nav1.2 (2KXW) because the structure included onlyCaMC). The solution structure of apo CaM bound to NaV1.5 (2L53)shows the ensemble of models included in the PDB deposit, withCaMN, having no preferred position relative to CaMC. In two crystallo-graphic structures (4DCK and 4OVN) of CaM bound to a fragment ofNaV1.5, there are distinct differences in the observed position of CaMN,

perhaps attributable to differences in crystal contacts, solution condi-tions, or an additional auxiliary protein (FGF13U/FHF2) bound to theNaV1.5 fragment in 4DCK (not shown). Residues in NaV1.5 and NaV1.6that make multiple contacts with CaM (spheres, Fig. 1D) include resi-dues in positions analogous to NaV1.2 Y1916 and Y1919 which are highlyconserved within NaV1.2 sequences.

Magnesium was resolved in the calcium-binding sites of two of thestructures of apo CaMbound toNaV1.5 (Fig. 1E). In 4OVN,Mg2+was ob-served in all 4 of the calcium-binding sites in CaM, whereas, in 4DCK,Mg2+ was seen only in CaMC sites III and IV (assignment of residuesin CaMN sites I and II was not complete in 4DCK). Magnesium andcalcium have different hydration shells, and chelation of magnesium isaccomplished with a different geometric signature in the subset of res-idues required for pentagonal, bipyramidal chelation characteristic ofcalcium binding to EF-hand loops [25] illustrated in SupplementalFig. S1A. In the crystallographic structure (3WFN) of apo CaM boundto NaV1.6 IQp, no metal ions were included in the deposited structure.

For NaV1.2, NaV1.5 andNaV1.6,WebLogos [26] in Fig. 1F illustrate thehighly similar sequences preceding and comprising their IQ motifs[IQRAϕRRϕxxK] (alignments are provided in Supplemental Table S1).All are basic, amphipathic alpha helices (BAA motifs) characteristic ofCaM binding domains. For example, the rat NaV1.2 IQ peptide has a cal-culated pI of 10.75, and a predicted net charge of+8 at pH 7. Consistentwith their similarity in sequence, the available high-resolution struc-tures (Fig. 1E) show that regardless of NaV isoform or structuralmethod(NMR, XRD) used for determination, the “IQ” residues are in identicalpositions. The I (cyan) of NaV1.2 and NaV1.5, and L (cyan) in the equiv-alent position of NaV1.6 are buried in the semi-open cleft of CaMC. Thefully conserved Q (green) residue of each IQ motif is oriented towardsthe F-G linker of CaM, forming a network of hydrogen bonds.

1.2. What does calcium binding do to CaM-NaV interactions?

While these analyses illustrate the conformation of CaM associatedwith the NaV IQ motif under low “resting calcium” conditions, they donot explain the role of calcium in modulating CaM-NaV interactions.Early studies identifying the preferred sites for CaM binding to theCTD of NaV1.2 showed that sites for apo CaM and (Ca2+)4-CaM wereoverlapping but slightly different [14], suggesting that (Ca2+)4-CaMmight slide towards the C-terminus of NaV1.2 upon binding calcium.

Calcium binding is known to trigger large changes in interhelical an-gles within both domains of CaM. In hundreds of high-resolution struc-tures of CaM –whether free or bound to a peptide derived from a targetprotein-calcium-free domains of CaM are always observed to adopt aclosed or semi-open conformation. The open tertiary structure is onlyobserved when calcium occupies the loops in the helix-loop-helix mo-tifs. This is illustrated in Fig. 2A, showing apo CaMC alone (1CFC) andbound to a myosin V peptide (2IX7), and calcium-saturated CaMC

alone (1CLL) and bound to a CaMKII peptide (1CDM). The alignmentby residues in helix G highlights the opening of the CaMC cleft in re-sponse to calcium binding; helices E and F are depicted in black. Thisgrid also demonstrates that the orientation of the CaMBD (CaM-bindingdomain) peptide frommyosin V bound to apo CaM is opposite to that ofthe CaMKII peptide bound to calcium-saturated CaMC. This plasticity inthe calcium-dependent interactions of CaMwith its targets is a hallmarkfeature of its role as a hub protein in signaling networks [27–32].

1.3. Distinct responses by CaMC and CaMN

The linker between CaMC and CaMN allows the two domains toadopt many different relative orientations. Full-length (Ca2+)4-CaMmay be extended as seen in the crystallographic structure in Fig. 2Bwhere the linker is helical, fully separating CaMN and CaMC. In contrast,Fig. 2C shows the two domains of (Ca2+)4-CaM wrapped around theCaMBD peptide of CaMKII (compact ellipsoidal). However, an overlay(Fig. 2D) of CaMC from Fig. 2B and C shows that CaMC adopts an opentertiary conformation in both, and has identical geometry of calcium-chelating residues in the calcium-binding sites, with the bidentateglutamates E104 (site III) and E140 (site IV), each at position 12 of anEF-hand loop (sticks in red or orange), providing two oxygens for hold-ing Ca2+ in the respective site. The geometry of the calcium-bindingloops and interhelical angles is very different if calcium is not bound.

The domains of CaM function as distinct calcium-sensors and CaMcan adopt a half-saturated state in which one domain is apo whileboth sites in the other domain are occupied by calcium. An example isthe structure of half-saturated CaM (having (Ca2+)2-CaMN and apoCaMC) bound to a fragment of the SK channel (1G4Y). This is comparedin Fig. 2E to a structure of apo CaM bound to a peptide from myosin V(2IX7). The apo CaMC domains are identical in tertiary structure, where-as CaMN has an open tertiary conformation with calcium bound in1G4Y. It adopts different positions relative to CaMC depending on thetarget peptide bound to CaM. Structures of apo CaMC bound to IQmotifsof NaVs (Fig. 1E) match those observed in 2IX7 and 1G4Y.

Two crystal structures of half-saturated CaMbound to the IQmotif ofNaV CTDs have been published inwhich calciumoccupies sites I and II inCaMN. For CaMbound to NaV1.5, Fig. 2F shows an overlay of the IQmotifof NaV1.5 bound to apo CaM (4DCK) and calcium-bound CaM (4JQ0). Inboth structures, CaMC is in the semi-open tertiary conformation charac-teristic of apo CaM, and the interface between CaMC and the NaV1.5 IQmotif is identical. Like the overlay shown in Fig. 2E, CaMN in 4JQ0 hasan open tertiary conformationwith calcium bound and adopts positionsrelative to CaMC that differ from apo CaMN in 4DCK. The gray spheresdepict the position of magnesium while the red depict calcium. Al-though both 4JPZ (3.02 Å resolution) and 4JQ0 (3.84 Å resolution)were deposited in the PDB with calcium having 100% occupancy insites III and IV of CaMC, the authors commented that “These conforma-tional differences of the individual CaM lobes in the NaV1.2/Ca2+ struc-ture suggested that the C-lobe was unlikely to be fully occupied,” (p. 4)and that CaMC “is essentially unoccupied in the NaV1.2/Ca2+ structure”based on anomalous scattering data (p. 6) [33].

1.4. Magnesium and calcium – cousins, not twins

The conclusion that sites III and IV do not contain calcium is support-ed by the overlay in Fig. 2G comparing CaMC in 4DCK and 4JQ0 (bothbound to the NaV1.5 IQ motif) and CaMC in 1G4Y (bound to an SK

3L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

Channel peptide). All three structures show CaMC having a semi-opentertiary conformation. The bidentate glutamates (E104 and E140) insites III and IV (blue 4DCK, cyan 1G4Y) are oriented away from theloop, consistent with the absence of calcium. These orientations arevery different from what is observed in calcium-saturated sites shownin Fig. 2D. In 4JQ0, only the alpha carbon of residues E104 and E140were assigned (the side chain atoms are missing). However the semi-open conformation, and absence of density for chelating carboxylgroups is consistent with the sites being apo.

The tertiary conformation of CaMC bound to the IQmotif in a frag-ment of NaV1.2 (4JPZ, Fig. 2H) is identical to that observed in 4JQ0representing NaV1.5. The conclusion that calcium is absent fromCaMC in 4JPZ is supported by the Fo-Fc electron density maps aroundthe metals in sites III and IV of chains C and I compared to the samesites in a CaM-alone structure (1CLL; Supplemental Fig. S1C). Despitethe high concentration of calcium in the crystallization buffer(300 mM sodium acetate, 50 mM Tris, pH 7.5, 2 mM CaCl2), all ofthese data suggest that CaMC maintained the apo state despitecalcium binding to CaMN. Thus, 4JPZ and 4JQ0 represent NaV CTD

fragments bound to a half-saturated complex of CaM like thecomplex observed in 1G4Y with an SK Channel fragment bound tohalf-saturated CaM (Fig. 2E).

However, calcium-saturated CaM is known to bind tightly to the IQmotifs in NaV1.2 and NaV1.5 [15,16,23,33–35]. A stoichiometric calciumtitration of CaM bound to the NaV1.2 IQ motif showed 4 equivalents ofcalcium were required for saturation ([16], Fig. 7 therein). This raisesthe question of why sites III and IV in CaMwould retain the apo confor-mation in structures 4JPZ and 4JQ0.

Those structures included a third protein – an intracellular FGF (FHF)isoform that recognizes the EFL domain within NaV. FGF13Uwas boundto the NaV1.2 CTD in 4JPZ, and FGF12U was bound to the NaV1.5 CTDin 4JQ0. For NaV1.2 (Fig. 2H), the asymmetric unit includes twoheterotrimers (called A and B), and there are many close interactionsbetween them. Close contacts between CaM in one heterotrimer withFGF in the other (i.e., CaMa-FGFb and CaMb-FGFa) are shown in Fig. 2I,while interactions between residues in the calcium-binding sites ofCaM in each heterotrimer are shown in Fig. 2J. These suggest that a net-work of contacts observed in the crystal structure may provide tertiary

Fig. 2. Ca2+-induced changes in calmodulin structure and linkage to target binding. A. Schematic showing structural changes of CaMC upon binding Ca2+ (horizontal) and a peptide target(vertical). Structures were aligned by CaM residues 117–128 (helix G). Interhelical angles for helices E and F (http://calcium.sci.yokohama-cu.ac.jp/efhand.html) shown in black are asfollows: apo CaMC (1CFC, 54.8°), apo CaMC bound to an IQ motif in myosin V (2IX7, 79.5°), (Ca2+)2-CaMC (1CLL, 89.7°), and (Ca2+)2-CaMC bound to CaMKII (1CDM, 88.5°). For 2IX7and 1CDM, the termini of the green target peptide are blue (amino) and red (carboxyl). B. (Ca2+)4-CaM in 1CLL. CaM residues 1–75 are blue, 76–80 are black, and 81–148 are red;Ca2+ ions are yellow. C. (Ca2+)4-CaM bound to CaMKII peptide in 1CDM. Color scheme the same as panel B, with peptide in green. D. Alignment of CaMC residues in B and C showingE104 and E140 in 1CLL (orange) and 1CDM (red) as sticks in Ca2+-binding sites. Label gives the average distance between carboxyl oxygens of E140 and the Ca2+ in site IV. CaMC ineach structure is shown in gray; structures were aligned using CaM residues 102–112 and 117–128 (helices F and G). E. Overlay of apo CaM bound to a myosin V IQ motif in 2IX7, andhalf-saturated CaM (CaMN Ca2+-saturated but CaMC apo) bound to an SK Channel peptide (1G4Y) aligned by CaMC helices F and G. F. Apo (Mg2+-bound) CaM and (Ca2+)4-CaMbound to NaV1.5 IQ motif (4DCK and 4JQ0). In sites III and IV, Mg2+ is gray, and Ca2+ ions listed in 4JQ0 are hot pink. Structures were aligned as in panel A. G. Alignment of apo CaMC

bound to SK Channel (1G4Y) shown in panel E, and apo (Mg2+ bound CaMC) bound to NaV1.5 (4DCK) and (Ca2+)4-CaM bound to NaV1.5 IQ motif (4JQ0) shown in panel F. Sidechains of E104 and E140 in 1G4Y (cyan) and 4DCK (blue) are shown as sticks; only backbone atoms for E104 and E140 were assigned in 4JQ0 (hot pink). Label gives the averagedistance between carboxyl oxygens of E140 and the Mg2+ ion in site IV in 4DCK. Mg2+ is gray, and Ca2+ assigned in 4JQ0 is hot pink. H. Dimer of heterotrimers (NaV-FGF13U-CaM) inthe 4JPZ crystal structure of (Ca2+)4-CaM bound to NaV1.2 CTD (residues 1777–1937, green) and FGF13U (purple). Circled regions correspond to regions circled in panels I and J. I.Blue circles highlight multiple contacts (≤6 Å) between CaM in one heterotrimer of 4JPZ with FGF13U in the other heterotrimer. Heterotrimers are labeled with subscripts “a” and “b”for tracking interactions. They are identical in sequence, and nearly identical structurally. J. Orange circle highlights highlight contacts (≤6 Å) between CaMC in one heterotrimer of4JPZ with CaMC in the other. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4 L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

constraints that favor retention of the CaMC-IQ binding interface ob-served in the absence of calcium.

1.5. Which CaM-NaV recognition determinants are encoded in the IQmotif?

Because there is no physiological evidence ofwhichwe are aware thatCaM interacts with FGF bound to a neighboring channel as seen in thedimer of heterotrimers in 4JPZ, it is expected that these interactionswould not occur under native conditions in a neuron. Based on the wellcharacterized effect of calcium triggering opening of hydrophobic cleftsin the domains of CaM [36,37], we hypothesize that calcium binding toCaMC bound to theNaV1.2 IQmotif should open the tertiary conformationof the domain to match the cleft observed for structures such as 1CLL or1CDM, shown in Fig. 2A. Furthermore, based on the original report ofCaMbinding sites inNaV1.2 [14] and subsequent titrations and thermody-namic linkage studies [7,15,16] showing differences in free energies ofbinding apo and (Ca2+)4-CaM, we expect calcium binding to alter theCaMC-IQ interface seen in apo-CaM-IQ complexes in Fig. 1E.

To compare how apo and (Ca2+)4-CaM differ in their recognition ofthe NaV1.2 IQ motif, we determined calcium-dependent differences inCaM binding to a biosensor comprised of the NaV1.2 IQ motif sequenceflanked by YFP and CFP [38]. The exquisite sensitivity of FRET betweenthe autofluorescent proteins allowed direct estimates of equilibriumconstants below 100 nM which allows us to distinguish between thevery high affinity of both apo and calcium-saturated CaM for the IQmotif.

To understand how calcium alters the interface observed betweenthe NaV1.2 IQ motif and apo CaMC, we focused exclusively on CaMC

bound to the IQ motif (no EFL) in the absence of auxiliary proteins(no FGF). We eliminated the potential contribution of crystal contactsby determining a high-resolution solution structure (2M5E) of thecomplex of (Ca2+)2-CaMC bound to Nav1.2IQp. In this structure,(Ca2+)2-CaMC reverses its orientation on the IQ motif relative to thestructure (2KXW) of apo CaMC bound to the same sequence. Thispivot in place is a striking structural response to calcium binding.

Our findings expand the repertoire of CaM gymnastics on ion chan-nels, and suggest a biophysical foundation for understanding an earlystep in themechanism of calcium-mediated NaV modulation. The avail-able crystallographic structures of half-saturated CaM bound to IQ mo-tifs from NaV1.2 (4JPZ) and NaV1.5 (4JQ0), and our solution studies ofcalcium-saturated CaMbound to the IQmotif of NaV1.2 suggest that cal-cium regulates a 3-step transition by binding to CaMN at intermediatecalcium levels, and at higher levels, binding to CaMC at the IQmotif, trig-gering its rotation around the IQ residues. The reversal of orientation ofCaMC would move CaMN at the least, and might require release and re-association, allowing CaMmolecules to interact elsewhere on NaV suchas at the inactivation gate ([39–44], other linkers within NaV1.2, or itsintracellular tails in amanner similar to CaM interactionswith CaV chan-nels [43–46]).

2. Materials and methods

2.1. Calmodulin

Full-length, wild-type CaM (paramecium sequence, 89% identical(131/148) to mammalian CaM) was overexpressed and purified asdescribed previously [15,47] with purity assessed by denaturingelectrophoresis, UV/vis spectroscopy and HPLC. Concentrations weredetermined using the BCA assay [48] (Pierce Biotechnology; Rockford,IL) and by absorbance of denatured CaM in 0.1 M NaOH (ε293.3 of2330 M−1 cm−1) [49].

2.2. YFP-CFP biosensors containing NaV1.2 IQ

A biosensor pET21B vector expressing a gene for YFP upstream of aKpnI site and CFP downstream of an AgeI site was a gift from A.Persechini andD.J. Black [38,50]. ADNAoligomer optimized for bacterial

expression and coding for the protein sequence of rat NaV1.2 residues1901 to 1927 (KRKQEEVSAIVIQRAYRRYLLKQKVKK) was purchasedfrom Integrated DNA Technology (Coralville, IA) and inserted betweenthe KpnI and AgeI sites (underlined residues constitute the canonicalIQ motif sequence, IQxxx[R,K]Gxxx[R,K]). Overexpression inBL21(DE3) cells was induced by addition of IPTG; cultures grew for40 h at 18 °C. Cells were lysed at 4 °C by sonication for 80 s in 50 mMHEPES, 100 mM KCl, 50 μM EGTA, 5 mM NTA, 1 mM MgCl2; pH 7.4and clarified by ultracentrifugation (Beckman TI-60, 25 K rpm, 45 min,4 °C). Biosensor expression was verified by SDS-PAGE; concentrationwas determined by UV–vis absorbance with ε514 of 83,400 M−1 cm−1

[51]. CaM binding to biosensor in clarified cell lysate was indistinguish-able from CaM binding to biosensor purified to homogeneity by nickelaffinity chromatography.

2.3. Calmodulin titrations of NaV1.2 IQ biosensors

The biosensor concentration ranged from 0.5 nM (for binding apoCaM) to 5 nM (for binding calcium-saturated CaM). Buffer was 50 mMHEPES, 100 mM KCl, 50 μM EGTA, 5 mM NTA, 1 mM MgCl2; pH 7.4,22 °C, with 1.5 μM BSA and 500 μMDTT. Calcium-saturating conditionsincluded 1 mM CaCl2. Titrations were conducted in a 1 cm Suprasilquartz cuvette (Hellma USA, Inc., Plainview, NY); the solution wasstirred continuously with a Teflon stir bar in a water-jacketed cuvette-holder held at 22 °C on a QuantaMaster-4 Steady State Spectrofluorom-eter System with a xenon lamp (Photon Technologies International;New Jersey, USA). The sample was excited at 430 nm and emissionspectra were collected from 450 to 550 nm. Peaks were observed at475 nm (CFP) and 525 (YFP) nm. An experimentally determinedisoemissive point (typically at 511 nm) was also monitored during thetitration. Bandpasses were 4 nm for excitation and 8 nm for emission;integration time was 4 to 8 s after each addition of CaM delivered bypositive displacement pipets (Drummond Wiretrol) from stocksrepresenting 10-fold serial dilutions spanning 6 orders of magnitude,from pM to μM. Normalized isotherms were determined from the frac-tional change in YFP intensity (525 nm) corrected for dilution (based onintensity at the isoemissivewavelength); the net decrease in YFP inten-sity (Fig. 3A) differed for binding apo and calcium-saturated CaM.

2.4. Free energy of CaM binding to NaV1.2IQ biosensors

Titrations were analyzed using nonlinear least squares analysis [52].The fractional saturation (Y) of the biosensorwas described by Eq. (1) inwhich Ka, the association constant, is the reciprocal of Kd, the dissocia-tion constant.

Y ¼ Ka " CaMfree½ $1þ Ka " CaMfree½ $ð Þ ð1Þ

To calculate [CaM]free, the independent variables [CaM]total and[biosensor]total were used in an iterative convergence algorithm accord-ing to the quadratic equation described in Eq. (2) in which b is (1+ Ka ·[biosensor] − Ka · [CaM]total).

CaMfree½ $ ¼−b(

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffib2−4Ka − CaMtotal½ $ð Þ

q

2Kað2Þ

Eq. (3) accounted for experimental variations in the observed endpoints of individual titration curves:

Signal ¼ f Xð Þ ¼ Y X½ $low þ Y " Span" #

ð3Þ

where Y refers to average fractional saturation of the biosensor andY[X]low corresponds to the intrinsic fluorescence intensity of biosensorin the absence of CaM. The variable Span represents the magnitude

5L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

Fig. 3. CaM binding to NaV1.2IQ biosensor and NaV1.2 IQp. Nav1.2IQ biosensor samples excited at 430 nm; peaks in emission intensity correspond to CFP (475 nm) and YFP (525 nm). A. Steady-state emission spectra of Nav1.2IQ biosensor alone (solidgreen) and saturatedwith apo CaM(black dashed) or calcium-saturated CaM(black solid) (N103-fold excess). B. Emission spectra of Nav1.2IQ biosensor alone (solid green) andwith N103-fold excess apo CaMN (blue solid) and calcium-saturated CaMN

(blue dashed). C. Equilibrium titrations of biosensor binding by apo full-length CaM (black dashed), CaMC (red dashed), and CaMN (blue dashed). D. Equilibrium titrations of biosensor binding by calcium-saturated full-length CaM (black solid), CaMC

(red solid), and CaMN (blue solid). Simulations in panels C and D are based on fits to the experimental data set shown in each panel. For CaMN, a horizontal line is provided to guide the eye. E. 15N-HSQC spectral overlay of (Ca2+)2-CaMC (red) and(Ca2+)4-CaM (black) bound to Nav1.2IQp (black). Backbone resonance assignments are shown for residues of peptide-bound CaMC. Green ellipses encircle resonances for the same residue in each complex. F. Expansion of crowded region of spectraloverlay in panel E. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

6L.H

oveyetal./BiophysicalChem

istry224

(2017)1–19

and direction of signal change upon titration (i.e., the difference be-tween the endpoints at high and low [CaM]). Following the signal offluorescence intensity of YFP at 525 nm, the Span was negative for in-creasing additions of CaM. The endpoints were fit directly in analysisof all titrations.

The quality of the nonlinear least squares fit was evaluated by judg-ing the square root of the variance, values of the 67% asymmetric confi-dence intervals, span and randomness of the distribution of residuals,and absolute values of elements of the correlation matrix [53]. Thesquare root of the residual variancewas typically b0.02. Themagnitudesof the confidence intervals were within a factor of two of the standarddeviation observed between independent, replicate titrations. Allaverage free energy values reported in Table 1 were based on 4 to 9replicate determinations from at least two preparations of biosensoroverexpressed from independent bacterial colonies. For CaMN, thedeflection of YFP intensity was so small at high [CaMN] that it was notpossible to estimate the affinity of CaMN for the biosensor.

The change in normalized fluorescence intensity of CaM or CaMC

binding to the biosensor was simulated in Fig. 3 using Eq. (3) thataccounted for the plateau intensity of free andCaM-saturated biosensor,as well as the decrease in intensity upon CaM binding. Free energies(and their corresponding equilibrium constants) are given in Table 1.Single-site isotherms of fractional saturation of the IQ motif by apo orcalcium-saturated CaM (Fig. 7A) were simulated according to Eq. (1)and the values in Table 1.

2.5. Simulation of calcium-binding isotherm

We previously measured energies of calcium binding to CaM aloneunder the conditions used in this study; in the absence of the IQ motif,∆G1 was−7.76 and ∆G2 was−15.92 kcal/mol [54]. Based on the prin-ciple of conservation of energy (shown in the linkage equations below),the difference in energy between apo CaM (∆Ga) and calcium-saturatedCaM (∆Gd) binding to the NaV1.2 IQ biosensor (Table 1, ∆∆G + 1.93)must be the same as the difference in calcium binding to CaM alone(∆Gc) and to CaM bound to the NaV1.2 IQ biosensor (∆Gb).

(Ca2+)4

CaMApo

CaM

Biosensor

(Ca2+)4 CaM Biosensor

4 Ca2+

( Gb) 4 Ca2+

( Gc)

Biosensor

( Ga)

Biosensor

( Gd)

CaMApo

ð4Þ

Thus, for illustration,we simulated a titration curve for calciumbind-ing to sites III and IV of CaM bound to the NaV1.2 IQ motif (in Fig. 7B)with a total free energy of−13.99 kcal/mol and the same cooperativityas observed for CaM alone.

2.6. NMR samples

Uniformly labeled 15N-CaMC or 13C,15N-CaMC was expressed inE. coli BL21(DE3) cells and contained a slight molar excess of commer-cially prepared peptide (GenScript, New Jersey). These representedNaV1.2 IQ motif residues 1901–1927, as previously described for NMRdetermination of the structure (2KXW) of the complex of Nav1.2IQpbound to apo CaMC [16]. In NMR samples, [CaM] was 1.5 mM in buffercomposition identical to that used previously [16] (10 mM imidazole,100 mM KCl, 0.01% NaN3, 50 μM EDTA, pH 6.8) in 90% H2O/10% D2Ofor amide-detected experiments or 100% D2O otherwise. Calcium inmatching bufferwas added to a final concentration of 3.3mM, to exceedthe concentration of sites (2 per complex).

2.7. NMR data acquisition

All experimentswere run on either a 500MHzBruker Avance II NMRspectrometer equipped with a TXI probe or an 800MHz Bruker AvanceII NMR spectrometer equipped with a TCI cryoprobe. All experimentsfor assigning both backbone and side chain resonances of the proteinwere collected at 500 MHz; experiments for assigning the unlabeledpeptide, as well as all NOESY experiments, were collected at 800 MHz.All NMR spectra were processed using NMRPipe [55] and analyzedusing both Sparky [56] and CCPN Analysis [57]. Chemical shiftdifferences were calculated on the basis of arithmetic differences inchemical shift (H or N) between the two spectra. ∆H and ∆N werescaled by the gyromagnetic ratios of 1H and 15N (normalized to 1H).The combined chemical shift difference, ∆δ, was calculated asffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiΔH2 þ ðΔN " 0:1013Þ2

q.

2.8. NMR resonance assignments

Backbone spectra (HNCA, HN(CO)CA, HNCACB, HN(CO)CACB,HNCO, and HN(CA)CO) for CaMC were analyzed in Sparky [56], andthe resulting peak lists were submitted to the PINE server [58] for auto-matic backbone assignment. The results were verified manually andcorrected as necessary in Sparky. Backbone assignments were thenimported into CCPN Analysis [57], which was used for all furtherassignments.

Side chain resonances were assigned using H(CCO)NH, C(CO)NH,and HCCH-TOCSY experiments, complemented by NOESY data for aro-matic rings and methionine epsilons. Unlabeled peptide was assignedusing 12C,14N filtered TOCSY (26 and 46 ms spinlock times) and12C,14N double filtered NOESY (80 and 120 ms mixing times) experi-ments to suppress signals from labeled protein [59–61]. 13C and 15NeditedNOESY spectra (120msmixing times), aswell as a 13C,15N edited,12C,14N filtered NOESY (140 ms mixing time, [62]), were peak pickedand initial assignments were made for unambiguous and somewhatambiguous crosspeaks (those with 3 or fewer possibilities).

Table 1Free energy of CaM binding to NaV1.2 IQ motif.

IQ Apo CaM Calcium-saturated CaM

∆GApoa Kd

b ∆∆Gc ∆G Ca2+ Kd ∆∆Gc ∆∆GdCa2+-Apo

CaM −11.48 ± 0.18 3.2 nM −9.55 ± 0.08 85 nM +1.93CaMC −10.72 ± 0.10 11.6 nM +0.76 −8.66 ± 0.09 386 nM +0.89 +2.06CaMN N100 μM N100 μM

Solution conditions: 50 mM HEPES, 100 mM KCl, 50 μM EGTA, 5 mM NTA, 1 mM MgCl2, 1.5 μM BSA, 500 μM DTT, ±1 mM CaCl2; pH 7.4, 22 °C.a Values of ∆G reported in kcal/mol. Averages based on 4 to 7 determinations from at least 2 independent preparations of biosensors, and two independent preparations of CaM.

Standard deviations from the mean are reported. Confidence intervals on independent trials ranged from ±0.04 to ±0.24 kcal/mol, with a median value of 0.10 kcal/mol.b Kd (equilibrium dissociation constant) calculated from average value of ∆G reported here.c ∆∆G = ∆GCaMc − ∆GCaM.d ∆∆GCa

2+-Apo = ∆GCa

2+ − ∆GApo.

7L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

Backbone and non-stereospecific side chain assignments of (Ca2+)2-CaMCwere straightforward and nearly complete (91% of all protons). Asindicated in Supplemental Table S2, 35 of 38 missing assignments arepossibly degenerate methylene protons, excluding M76 for which onlythe epsilon methyl is observed. Assignment of the residues for unla-beled Nav1.2IQp was more difficult due to chemical shift degeneracyand the lack of an isotopic label. No assignments were obtained forthree residues at each end of the peptide (1901–1903 and 1925–1927). The remainder of the peptide, which contained all residues thatcontact CaMC, was assigned with sufficient completeness for structuredetermination (77% of expected protons); 19 of 33 missing peptide as-signments may have degenerate chemical shifts and many are in sol-vent-exposed side chains (Supplemental Table S3).

2.9. Structure calculations

Initial restraints for structure calculations were generated in CCPNAnalysis [57]. Distance restraints were made for all assigned NOEs,and shift matchingwas used to generate additional ambiguous distancerestraints. NOEswere grouped into bins based onmeasured peak inten-sities. Dihedral angle restraints were generated for both the protein andpeptide using the built in DANGLE routine [63]; a residue-by-residuecomparison showed that average values for the protein were highlyconsistent with those determined using TALOS+ [64]. Additionally, aset of hydrogen bond restraints was generated to constrain alphahelices.

High-resolution models were generated using traditional solutionNMR techniques of combining distance constraints derived fromNOESY spectra with backbone φ and ψ dihedral angles derived fromchemical shifts. Initial rounds of structure determination were carriedout with Aria/CNS [65,66] using standard protocols and importing re-straints directly from CCPN Analysis [57]. Intermediate structureswere generated in Xplor-NIH [67,68] or CNS [66]. The results wereused to manually curate restraint tables in an iterative manner. Addi-tional hydrogen bond restraints were included to constrain residues inalpha helices. To verify the validity of these restraints, an ensemble cal-culated with the hydrogen bond restraints removed yielded a some-what lower resolution but otherwise similar structure, suggesting thatthis data is not significantly biasing the final result (data not shown).After multiple rounds of structure calculations, restraint and violationanalysis suggested no further changes to the distance restraints. Thefinal ensemble of structures was generated as follows.

An ensemble of 300 structures was generated using CNS [66] froman extended starting structure of calcium-free CaMC using torsionangle dynamics, out of which the 20 with the lowest energies wereretained. This stage used the default anneal.inp script and consisted ofa brief initial minimization followed by 1000 steps of high temperaturedynamics at 50,000 K, 1000 steps of cooling from 2000 K to 0 K, and fi-nally 2000 steps of minimization. For the 20 lowest energy structuresfrom this calculation, calcium ions were then added and placed veryroughly in the calcium-binding loops. An additional set of restraintsfor positioning the calcium ions was derived from a crystal structureof calcium-saturated CaM (1EXR.pdb) [69] and included in a furtherCNS [66] refinement protocol using Cartesian dynamics and consistingof 2000 steps at 2000 K, 2000 cooling steps from 1000 K to 0 K, and fi-nally 2000 steps of minimization. The final force constants used were50 kcal/mol Å−2 for distance restraints and 200 kcal/mol rad−2 for tor-sion angle restraints. CNS [66] reported no backbone dihedral angle vi-olations N2° and no distance violations N0.2 Å in this final ensemble.Complete structure statistics are reported in Table 2.

The final structures have good Ramachandran statistics as deter-mined by PROCHECK-NMR [70] (Table 2 and Supplemental Fig. S2).For the entire ensemble of 20 structures, 99.6% of non-glycine residuesfall in the most favorable or allowed regions of torsion angle space(100% for the minimized average structure). All residues falling in thegenerously allowed (0.35%) and disallowed (0.05%) regions correspond

to either Q1904 or K1924 which are only partially assigned and are at thedisordered ends of the peptide. The PROCHECK-NMR [70] analysis omit-ted residues 76–78 and 148 in CaMC, which were poorly ordered, andresidues 1901–1903 and 1925–1927 in Nav1.2IQp, for which no NMRdata or restraints are available.

All molecular models were drawn with PyMOLMolecular Visualiza-tion System v. 1.7.2.3 (Schrödinger, LLC).

3. Results

3.1. FRET intensity of CaM-biosensor assemblies

To evaluate separable roles of CaM domains in recognition of theNaV1.2 IQ motif, and to estimate equilibrium binding constants forboth apo and calcium-saturated CaM and its domains, we monitoredCaM-induced disruption of FRET in a biosensor protein [38,50] thatwas engineered to contain the desired NaV1.2 sequence (residues1901–1927) bracketed by YFP and CFP (see Materials and methods).

Table 2Structural statistics for 2M5E for the final ensemble (20 lowest energy structures).

Experimental restraints

CaM NOEsa

Intraresidue (i = j) 492Sequential (|i − j | = 1) 393Medium range (1 b | i − j | b 5) 415Long range (|i − j | ≥ 5) 399Total CaM NOEs 1699

Peptide NOEs 279Intermolecular NOEs 97Total unambiguous NOEs 2075NOEs with multiple assignments 651Total NOEs 2726Distance restraints per residue, CaMa 23.6Distance restraints per residue, peptideb 13.3phi/psi angles, CaM 136phi/psi angles, peptide 38

Restraint violations Ensemble Minimized average

NOE distances violated N0.2 Å 0 0Dihedral angles violated N2° 0 0

RMSDs from experimental restraintsNOE (Å) 0.0039 ± 0.0001 0.004Dihedrals (°) 0.068 ± 0.021 0.082

XPLOR energies (kcal/mol)Overall energy 50.63 ± 1.33 54.93Bonds 0.74 ± 0.06 1.00Angles 41.10 ± 0.28 41.92Impropers 1.02 ± 0.08 1.09van der Waals 5.50 ± 0.96 8.67NOEs 2.21 ± 0.12 2.17Dihedrals 0.05 ± 0.03 0.07

RMSD from idealized geometryBond lengths (Å) 0.0007 ± 0.0001 0.0008Bond angles (°) 0.300 ± 0.001 0.303Impropers (°) 0.090 ± 0.003 0.093

Ramachandran plot statistics (%)c

Most favored regions 92.0 90.6Additionally allowed regions 7.6 9.4Generously allowed regions 0.4 0.0Disallowed regions 0.1 0.0

Coordinate RMSD (average difference to mean, Å)c

Backbone atoms 0.643Heavy atoms 1.188

Restraint violations, RMSDs and energies are for all residues in the construct. Structuralstatistics are taken from the output of CNS [66], with the exception of the Ramachandranstatistics which come from PROCHECK-NMR [70].

a For CaM residues 77–148 (all except M76).b For peptide residues 1904–1924 (all assigned).c For structured residues (CaM residues 79–147, peptide residues 1904–1924).

8 L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

Steady-state emission spectra (λex = 430 nm) for the NaV1.2IQ biosen-sor alone (green, 4 nM)with λmax at 475 nm for CFP and 525 nm for YFPare shown in Fig. 3A.

FRET intensity is sensitive to small changes in distance betweenfluorophores induced by CaM binding to the IQ motif sequence in theNav1.2IQ biosensor. We have shown previously that the IQ motifsequence is intrinsically disordered [15]. This allows CFP and YFP to bein close proximity for efficient non-radiative transfer of energy. Thetypical change in intensity of the biosensor after saturation with apo(black, dashed) or calcium-bound (black, solid) CaM is shown inFig. 3A ([CaM]:[biosensor] = 103:1). The emission spectra werebuffer-subtracted and corrected for dilution; the spectra were normal-ized to the peak intensity of free biosensor at 525 nm. The intrinsicfluorescence of CaM (which contains Phe and Tyr, but not Trp) doesnot contribute measurably to the intensities observed.

At λmax for YFP, the fractional decrease in intensity was consistentlylarger for saturation by apo CaM (36 ± 1%) than (Ca2+)4-CaM (21 ±1%). We infer that apo CaM and (Ca2+)4-CaM differ in conformationwhen bound to the IQ motif, and thus differentially reduce energytransfer from CFP to YFP by changing their relative orientation or spatialseparation. The isoemissive point for free and CaM-saturated biosensorwas observed consistently at 511 nm throughout the titration, indicat-ing that it behaved as a two-component solution.

3.2. Domain-specific binding of CaM

To investigate whether both domains of CaMwere necessary for thechange influorescence intensity seenupon binding full-lengthCaM, a C-domain fragment (CaMC, residues 76–148) or an N-domain fragment(CaMN, residues 1–75) was added to the Nav1.2IQ biosensor (4 nM) inlarge excess ([CaM]:[biosensor] = 103:1). The emission spectra of theNav1.2IQ biosensor with apo and calcium-saturated CaMC bound (datanot shown) were very similar to those in Fig. 3A for full-length CaM.However, Fig. 3B showed a negligible change (~1%) in intensity of theNav1.2IQ biosensor after addition of apo CaMN (dashed blue), and a netchange of ~3% for calcium-saturated CaMN (solid blue) at a concentra-tion of 15 μM CaMN. The deflection in fluorescent intensity at 50 μMCaMN was b1% of that seen at 50 nM CaMC, showing high specificityfor CaMC. These results are consistent with our prior studies using fluo-rescence anisotropy to study CaM and its domains binding to a fluores-cein-labeled peptide corresponding to the same Nav1.2 IQ motifsequence [[15,16].]

3.3. Energetics of CaM binding

Thermodynamic linkage analysis previously showed that apo CaMbinds more favorably than calcium-saturated CaM to rat Nav1.2IQp[14–16]. However, the free energies of binding could not be determinedquantitatively because the high concentration of Nav1.2IQp needed toobtain sufficient signal in fluorescence anisotropy or gel electrophoresismobility shift experiments resulted in stoichiometric titrations. Incontrast, the strongfluorescence intensity of theNav1.2IQ biosensor per-mitted titrations to be conducted at concentrations as low as 0.5 nM(see Materials and methods). The fractional change in intensity atλmax for YFP (normalized to biosensor alone at 100% and CaM-saturatedbiosensor as zero) was plotted as a function of total concentration ofadded CaM or CaMC, under either apo (Fig. 3C), or high calcium(1 mM, Fig. 3D) conditions.

These binding isotherms alloweddirect estimates of Gibbs free ener-gies of binding (∆G) and corresponding dissociation constants (Kd)under equilibrium conditions (Table 1). Apo CaM binding to theNav1.2IQ biosensor (−11.48 kcal/mol (Kd 3.2 nM)) was more favorableby−1.93 kcal/mol than the binding of calcium-saturated CaM (−9.55kcal/mol; Kd 85 nM). The difference was significantly larger than thestandard deviations (0.18 and 0.08 kcal/mol) of multiple replicate titra-tions for apo and calcium-saturated CaM, respectively. The difference

similarly exceeded the confidence intervals for individual determina-tions (median value of 0.10 kcal/mol), and suggested that the interfacebetween apo CaM and the IQ motif changed when sites III and IV filledwith calcium.

Because CaMN showed negligible binding to the Nav1.2IQ biosensor(Fig. 3B), it was not possible to observe a titration or estimate a free en-ergy of interaction. However, to visually compare its behavior to that ofCaMand CaMC under apo (Fig. 3C), or high calcium (1mM, Fig. 3D) con-ditions, the small change in intensity observed upon addition of CaMN

was normalized by assuming that the YFP signal of the biosensorwould undergo the same deflection as observed for CaM (i.e., 36% forapo and 21% for calcium-saturated CaM) if it were saturated by CaMN.Even if the slight deflection observed at 10 μM CaMN were attributableto specific binding rather than non-specific quenching, the midpointof a complete titration would be higher than 100 μM, correspondingto a very weak dissociation constant. It is also possible that this modestbinding represents CaMN binding to the same NaV sequence that is oc-cupied by CaMC when full-length CaM is bound, and thus representsan interaction that would not be populated in the cell where only full-length CaM is available. We observed a similar phenomenon for do-main-specific binding of CaM to melittin [71].

3.4. CaMC binding to Nav1.2IQp independent of CaMN

The titrations in Fig. 3 indicated that the driving force for CaM bind-ing to the IQ motif comes primarily from residues in the C-domain ofCaM. Although the binding of CaMC was slightly less favorable thanCaM for each condition (see Table 1), this may reflect electrostatic dif-ferences in attraction for the very basic Nav1.2IQ sequence, rather thandifferences in close contacts such as van der Waals interactions or hy-drogen bonding at the interface of CaM and Nav1.2IQp. The calculatednet charge at pH 7 (http://www.scripps.edu/~cdputnam/protcalc.html) of CaM is −23.5 and CaMC is −12.6. Approximating CaM andCaMC as negative point charges binding to positive IQ motifs, theCoulombic contributions (proportional to q1 ∗ q2/r2 [72]) to theirfree energies of binding to the IQ motif would differ by the squareof the ratio of their charges (23.5 vs. 12.6), or a factor of ~4. Thatrepresents a free energy of −RTln(4) or 0.813 kcal/mol at 22 °Cwhich corresponds well with the ∆∆G values of 0.76 and 0.89 kcal/molreported in Table 1.

To assess directly whether the CaM-peptide interface was the samefor (Ca2+)4-CaM-Nav1.2IQp and (Ca2+)2-CaMC-Nav1.2IQp, HSQC spectraof 13C,15N-labeled complexes of (Ca2+)4-CaM (black) and (Ca2+)2-CaMC (red) bound to Nav1.2IQp were compared (Fig. 3E). The greenellipses encircle resonances for the same residue in each complex, andshow that they are in identical or nearly identical positions, indicatingthat their chemical environment is very similar. Resonances in thehighly overlapped central region boxed by a dashed line are shown inan enlarged format in Fig. 3F.

Consistent with the interface between Nav1.2IQp and CaM beingdominated by close contacts with CaMC, resonances within the N-domain of (Ca2+)4-CaM bound to the Nav1.2IQ peptide matched thosein a free fragment (Ca2+)2-CaMN (see Supplemental Fig. S2). Thesestudies match prior NMR studies showing that CaM binding Nav1.2IQpcaused few perturbations of residues within the N-domain of eitherapo or calcium-saturated CaM [16].

Therefore, to determine why (Ca2+)2-CaMC binds less favorablythan apo CaMC to the NaV1.2 IQ motif, and understand how calciumbinding changes the interface between CaMC and the NaV1.2 IQ motifwithout the influence of auxiliary proteins or flanking regions, we de-termined a high resolution structure of CaMC bound to a peptiderepresenting the IQ motif (hereafter referred to as the CaMC-Nav1.2IQpcomplex). NMR allowed determination of the complex in solutionwith-out packing constraints that might be contributed by a crystallographiclattice.

9L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

3.5. Solution structure of Ca2+-CaMC bound to Nav1.2IQp

The structure was determined using a sample of 13C,15N-(Ca2+)2-CaMC bound to unlabeledNav1.2IQp (seeMaterials andmethods). Nearlycomplete assignments of CaMC and central Nav1.2IQp peptide residuesthat interact with CaMC were obtained (see Supplemental Tables S2and S3). Backbone traces of the 20 NMR-derived lowest energy struc-tures of the complex are superimposed in Fig. 4A in two orientations:with Nav1.2IQp perpendicular and parallel to the plane of the figure.The Ramachandran plot is in Supplemental Fig. S3; Table 2 providesstructural statistics. In the minimized average NMR structure of thecomplex (Fig. 4B), (Ca2+)2-CaMC adopts a 4-helix bundle in the classicopen conformation (see Fig. 2A), with side chains in the EF-hand sitesconsistent with the metal-coordination geometry adopted when calci-um is bound (see Supplemental Fig. S1B). Residues 1904–1923 ofNav1.2IQp form an amphipathic α-helix closely associated with(Ca2+)2-CaMC. The terminal residues (1901–1903, 1924–1927) ofNav1.2IQp were disordered, as had been observed for Nav1.2IQp boundto apo CaMC.

Analysis of the minimized average structure with CSU (Contacts ofStructural Units [73]) in Fig. 4C showed residues in Nav1.2IQp (bottomrow) that are in the binding interface with CaMC, and the vertical stacksshow CaMC residues that have atoms within 6 Å of Nav1.2IQp; contactsthat were ≤4.5 Å are shaded orange.

The side chainsV1911, I1912, A1915 and Y1916 (Fig. 4B)makemany con-tacts with the hydrophobic cleft of CaMC, including the FLMM residues(F92, L112, M124, M144) observed to bind a large number of peptidesin calcium-saturated CaMC complexes ([74–76]). Residue Q1913 in theIQ motif of Nav1.2IQp points towards the C-terminus of CaMC.

For determining the orientation of CaMC relative to the peptide, thekey intermolecular NOEs observed between CaMC and Nav1.2IQp werethose associated with CaMC residues A88, V91 and M124 (Fig. 4D).Nav1.2IQp has only two aromatic residues (Y1916 and Y1919) with distinctchemical shifts, each of which makes unambiguous NOE contacts toboth A88 and V91. The side chain of A88 comes within 2.4 Å of Y1916

(vs. 7.8 Å in 2KXW), and the V91 side chain is within 3 Å of Y1919 andjust over 5 Å from Y1916 (both N10 Å in 2KXW). Furthermore, the sidechain of M124 makes contacts with Hα and both Hβs of S1908, which isthe only serine in Nav1.2IQp and N11 Å away from M124 in the corre-sponding apo structure (2KXW).

To see the overall effect of peptide binding on (Ca2+)2-CaMC, a1H-15 N HSQC of (Ca2+)2-CaMC bound to Nav1.2IQp was compared to acorresponding spectrum of (Ca2+)2-CaMC alone (Fig. 5A). The chemicalshift perturbations observed for each backbone amide upon binding toNav1.2IQp localized mainly in helices E, F, and H (Fig. 5B) and the largestdifferences in chemical shift were in regions contacting Nav1.2IQ (Fig.5C). The resonances for residueswithin sites III and IV in these two sam-ples were nearly identical regardless of the peptide binding, suggestingthat the geometry of calcium-chelation is also identical. As noted inMaterials and methods, NMR does not directly visualize the positionof metal ions. Therefore, the positions of calcium ions shown in Fig. 5Cwere fit based on the side chain geometry of highly acidic coordinatingresidues. Such residues would undergo electrostatic repulsion if therewere no divalent cation present.

3.6. Reversal of CaMC on the IQ motif

The most striking feature of the solution structure (2M5E) of calci-um-saturated CaMC bound to Nav1.2IQp was the orientation of(Ca2+)2-CaMC relative to Nav1.2IQp. According to the NOEs shown inFig. 4D, it was opposite to that observed in apo CaMC-Nav1.2IQp in2KXW [16]. A nearly 180° rotation of a CaM domain on the same hydro-phobic face of a target CaMBD helix was an unexpected response. Theprotein samples used for determining 2M5E were made by adding ex-cess calcium to an equilibrated complex of apo CaMC bound to Nav1.2IQpthat was from the same stock used for determining 2KXW (see

Materials and methods). Thus, the final structure shown in Fig. 4 re-quired that (Ca2+)2-CaMC either pivoted in place, or released and re-as-sociated with Nav1.2IQ.

In both 2M5E and 2KXW, the sequence of Nav1.2IQ was sufficientlylong to encompass residues that interact closely with CaMC as well ascontaining terminal residues (1901–1904 and 1924–1927) that werenon-interacting and highly disordered. Thus, theNav1.2IQp sequence ap-peared to bracket residues needed for tight binding to CaMC. The com-parison of HSQC spectra of (Ca2+)2-CaMC and apo CaMC, each incomplex with unlabeled Nav1.2IQp (Fig. 6A), showed that few CaM res-onances overlapped. A comparison of 12C,14N filtered NOESY spectrafor both apo and calcium-saturated CaMC-Nav1.2IQp complexes (partialspectra shown in Supplemental Fig. S4) shows striking differences con-sistentwith calciumbinding inducing amajor conformational change asrepresented in Figs. 4 and 5.

Some changes in peak positions reflect the expected effects of calci-um occupancy of sites and the opening of the tertiary structure trig-gered by calcium binding (i.e., semi-open to open conformation) thatwould occur for CaM alone. However, spectral comparisons of calci-um-saturated CaMC ± Nav1.2IQp in Fig. 5A showed that binding of thepeptide made additional changes in CaM peaks beyond those causedby calcium binding. The magnitude and direction of the perturbationsseen in Fig. 6A reflect binding of calcium at sites III and IV, and bindingof Nav1.2IQp in the open cleft of CaMC.

The chemical shift perturbation for each resonance that wasassigned in both 2KXWand 2M5E (Fig. 6B) showedmany large changesbetween the structures (average∆δ of 0.5 ppm).With a false-color scaleof red (representing greatest) to blue (least) perturbation, Fig. 6C high-lights that themajor differences outside calcium-binding sites III and IVwere in the FG-linker which changes contacts dramatically when CaMC

pivots relative toNav1.2IQ. Consistentwith analysis of short-range struc-tural contacts (Fig. 4C) in (Ca2+)2-CaMC (2M5E), residues in the CaMC

FG-linker contact A1915 in Nav1.2IQp whereas in apo CaMC (2KXW), theFG-linker made a network of hydrogen bonds with Q1913. Because ofthe reversal of (Ca2+)2-CaMC onNav1.2IQp, Q1913 in 2M5Eprimarily con-tacts residues near the C-terminus of CaM and the residues that wouldbe closest to CaMN.

Fig. 6D and E (90° rotations) shows 2M5Eand 2KXWaligned accord-ing to the backbone of Nav1.2IQp. Positions of selected side chains (I1912,Q1913, A1915, Y1916 and Y1919) that make multiple contacts with CaM areshown in ball-and-stick format. CaMC residue 76 (blue) indicates thejunction with CaMN, highlighting the reversal of CaM relative to thechannel peptide. The open conformation of (Ca2+)2-CaMC (2M5E)(Fig. 6D) has amore shallow hydrophobic cleft than the semi-open con-formation of apo CaMC (2KXW). A view looking into the clefts (Fig. 6E)shows that in apo CaMC (2KXW), the side chains of I1912, Q1913, A1915,Y1916 are tightly clustered in the deep semi-open cleft whereas theseresidues are distributed over a wider surface of the hydrophobic cleftin 2M5E, with Y1916 oriented closer to Y1919 in the calcium-saturatedstructure.

Although the rotation of (Ca2+)2-CaMC on Nav1.2IQp was unexpect-ed, the observed polarity of Nav1.2IQp relative to CaMC is consistentwith hundreds of other complexes of calcium-saturated CaM bound topeptides representing CaMBDs from other ion channels, receptors andenzymes (see Fig. 2A). These motifs are commonly denoted by a num-bering scheme (such as 1-5-8) indicating the positions of residues inthe primary sequence that interact closely with CaM clefts. In 2M5E,Nav1.2IQp residues I1912-Y1916-Y1919 correspond to positions 1-5-8 in atypical motif, and I1912 is the sole residue contacting all 4 of the FLMMresidues [75] in CaMC.

4. Discussion

CaM is an essential, intracellular, eukaryotic calcium sensor that reg-ulates both aqueous and membrane-spanning target proteins. In turn,those targets are allosteric effectors of the calcium-binding free energies

10 L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

Fig. 4.NMRmodels of Ca2+-saturated CaMC bound to NaV1.2IQp. A.Wireframe backbone representations of the 20 lowest energy structures and the ensembleminimized averagemodel of(Ca2+)2-CaMC bound to Nav1.2IQp (2M5E). For clarity, only well ordered helical residues 1904–1923 of Nav1.2IQp are shown. B. Cartoon representation of minimized average model in2M5E. Nav1.2IQp helix shown as a ribbon with gradient from blue (N-terminus) to magenta (C-terminus). Cylinders represent CaM helices E (gray), F (red), G (orange) and H (gray)with yellow spheres for calcium ions. Residues of the IQ motif making ≥3 short-range contacts (b4.5 Å) with the hydrophobic cleft of CaM are shown in ball-and-stick representationalong with Q1913 for reference. C. Analysis by CSU (Contacts of Structural Units) [73] of the minimized average model of (Ca2+)2-CaMC-Nav1.2IQp. The primary sequence of residues1904 to 1923 of the IQ motif is shown on the x-axis (I1912 in cyan, Q1913 in green). Boxes listing residues of CaM located within 4.5 Å of each IQ motif residue are shaded in orange;longer range contacts (4.5 to 6 Å) are in white boxes. D. NMR strip plots corresponding to three CaM residues: A88α, V91γ2 and M124ε. For each residue, the top panel represents a13C-edited, 12C,14N-filtered NOESY experiment, the middle panel represents an HCCH-TOCSY experiment, and the bottom panel is a 13C-edited NOESY with carbon chemical shift ofthe planes denoted in the label. Labels for atoms in Nav1.2IQp are green and in CaM are red. Residues surrounding A88, V91 and M124 of CaM are shown in images made with PyMOL(Schrödinger, LLC) next to the strip plots. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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of CaM,making CaM an effectivemodulator over a range of intracellularcalcium concentrations that spans at least three orders of magnitude,bracketing its micromolar affinity for calcium as a free protein [77,78].Voltage-dependent sodium channel NaV1.2 retains CaM as a constitu-tive subunit, but the mechanism of calcium-triggered change in thecomplex has been perplexing because previous structural studies didnot see calcium bound to CaMC which anchors CaM to the channel.

4.1. All IQs are BAAs, but not all IQs are equal

The magnitude and direction of a target-linked shift in calcium-binding affinity of CaM and its conformational response when boundto a target is not predictable from the CaMBD sequence alone. Allknown CaMBDs are BAAs - Basic sequences (pI of 10–12) that form Am-phipathic Alpha helices (usually continuous). Many bind exclusively tothe open conformation of domains in (Ca2+)4-CaM,with nomeasurableaffinity for apo CaM. Others, such as the NMDA receptor [79] and phos-phodiesterase [80], retain apo CaM as a constitutive subunit but have ahigher affinity for calcium-saturated CaM than apo CaM which bindsprimarily via contacts with CaMC. Calcium binding to CaMN changesthe mode of interaction of CaM with the target protein.

The set of target proteins that binds the semi-open cleft of apo CaMdomains more favorably than the open cleft of calcium-saturated CaMdomains include neuromodulin [81–83] and neurogranin [82,84] aswell as some myosins. Their CaMBDs are IQ motifs, a subclass of BAAmotifs, and some release CaM when calcium binds CaM [85]. Sequencevariations flanking the IQ motif influence whether only CaMC, or bothdomains of CaM, interact with the IQ motif [27,86–88].

For the well conserved IQ motifs in human voltage-gated sodiumchannels, NMR and crystallographic studies of CaM binding to NaV1.2,

1.5, and 1.6 have shown that apo CaMC anchored CaM on the IQ motif,while apo CaMN adopted variable positions and made few contactswith the channel fragment. This was consistent with thermodynamicdata that showed undetectable or very weak binding to these IQ motifsby CaMN. Through linkage or direct thermodynamic measurements,these studies also showed that the NaV IQ motifs have a higher affinityfor apo CaM than (Ca2+)4-CaM [15,16,22,33,41]. These studies of NaV-CaM interactions contrast with the behavior of IQ motifs in voltage-de-pendent calcium channels such as CaV1.2, which interactswith both do-mains of CaM and has a higher affinity for (Ca2+)4-CaM than apo CaM[89–91], despite the similarities between the calcium and sodium chan-nels [92].

4.2. Apo and (Ca2+)4-CaM binding to NaV1.2 IQ

The IQ-motif of NaV1.2 binds both apo CaM and (Ca2+)4-CaM withhigh affinity (Table 1). In our earlier studies of CaM binding to afluoresceinatedpeptide of theNaV1.2 IQmotif, the stoichiometric condi-tions required for signal-to-noise ratios that yielded reproducible mea-surements allowed us only to put limits on the dissociation constantsbecause the Kd was similar to, or much lower than, the concentrationof peptide being titrated. This challenge of estimating dissociation con-stants plagues many biophysical methods such as quantitative mobilityshift assays and isothermal titration calorimetry where it can be impos-sible to detect a concentration of macromolecule that is an order ofmagnitude lower than the value of the Kd, leading researchers to con-duct studies under stoichiometric conditions.

To address the low spectral intensity inherent in our prior studiesusingfluorescence anisotropy, herewe employed the biosensormethodof Black and Persechini [38]. The high steady-state intensity of YFP-CFP

Fig. 5. Chemical shift differences and contacts in (Ca2+)2-CaMC ± Nav1.2IQp. A. HSQC spectral overlay of (Ca2+)2-CaMC alone (red) and bound to Nav1.2IQp (black). Backbone resonanceassignments are shown for residues of peptide-bound CaMC. B. Effects of Nav1.2IQp on chemical shifts of (Ca2+)2-CaMC. Changes in chemical shifts are calculated as described inMaterials and methods. Location of CaM helices and calcium-binding loops are shown above the plot. Residues in calcium-binding sites III (93–104) and IV (128–140) are indicated byyellow shading. C. Chemical shift perturbations mapped onto the structure of (Ca2+)2-CaMC in 2M5E. The greatest effect of Nav1.2IQp binding is indicated by the red/wide cartoon, andthe weakest effect is shown by the blue/narrow cartoon. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

12 L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

FRET allowed titrations to be conducted biosensor concentrations aslow as 0.3 nMbiosensor, a factor of 10 lower than themost favorable ob-served Kd. Although it would have been preferable to use an even lowerconcentration of biosensor, in the nonlinear least squares analysis to de-termine free energies, the difference between free and total [CaM] wasaccounted for by using a function that calculated free [CaM] from thetotal [CaM] and the degree of saturation (see Materials and methods).

Simulations of saturation of the NaV1.2 IQ motif based on the ∆Gvalues reported in Table 1 for apo (dashed) and calcium-saturated(solid) CaM are shown in Fig. 7A. The 30-fold separation in midpointsindicates that the CaM-IQ interface must be different, but a small con-formational change that resulted in the loss of a few hydrogen bondsmight account for this weakening of affinity for (Ca2+)4-CaM. Instead,our solution structures 2KXW and 2M5E (insets in Fig. 7A) showed

Fig. 6. Comparison of nested anti-parallel sites in Nav1.2IQp. A. HSQC overlay of CaMC-Nav1.2IQp with (black) andwithout (green) saturating calcium. Backbone resonance assignments areshown for residues of (Ca2+)2-CaM-Nav1.2IQp. B. Chemical shift differences between CaMC-Nav1.2IQp ± Ca2+ indicated changes in chemical environment of individual CaM residues.Location of CaM helices and calcium-binding loops are shown above the plot. Residues in calcium-binding sites III (93–104) and IV (128–140) are indicated by yellow shading. C.Chemical shift perturbations mapped onto the structure of (Ca2+)2-CaMC in 2M5E. The greatest effect of Ca2+ binding is shown in the cartoon as red/wide, while the weakest effect isblue/narrow. D. Comparison of (Ca2+)2-CaMC (2M5E) and apo CaMC (2KXW) bound to Nav1.2IQp aligned by superposition of the helical backbone of Nav1.2IQp. Residues I1912 (cyan),Q1913 (green), and A1915 (black) are in ball-and-stick. For clarity, the peptide backbone is omitted. The surface of solvent-exposed CaM residues is red and cleft residues are orange insemi-open (2KXW) and open (2M5E) CaM. CaMN would connect to CaMC via residue M76 (blue) highlighting the reversed orientations of CaMC on the IQ motif. E. Comparison ofclefts of Ca2+-CaMC (2M5E) and apo CaMC (2KXW) binding Nav1.2IQp. Structures were aligned based on helical residues of the peptide. IQ motif residues I1912 (cyan) Q1913 (green),A1915 (black), Y1916 (magenta), and Y1919 (dark green) are shown in ball-and-stick representation. For clarity, the backbone of the peptide is not shown. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7.Models of CaM retention, release, or reversal onNaV1.2. A. Simulations of saturation of theNaV1.2 IQmotif by apo (dashed) and calcium-saturated (solid) CaMbased on∆G values inTable 1.Molecularmodels show calcium-induced reversal of CaMC relative to the IQmotif peptide. B. Simulation of calciumbinding to CaMbound to theNaV1.2 IQmotif (seeMaterials andmethods). Resting calcium levels indicated by blue bar; green bar centered on 10 μM indicates range of neuronal intracellular Ca2+ levels sufficient to achieve 50–75% calcium-saturatedCaMC bound to NaV1.2-IQ. Schematics indicate calcium-induced reversal of CaMC on the IQ motif. C. Schematic for proposed 3-state mechanism of CaM-mediated regulation based oncalcium titration of full-length CaM bound to NaV1.2IQp monitored by NMR. Apo CaM binds to the IQ motif via CaMC with no preferred contacts between CaMN and Nav1.2IQp. Calciumbinding to sites I and II affects only CaMN; calcium binding to sites III and IV triggers reversal of CaMC on the IQ motif. D. CaM-target structures representative of the 3 states shownschematically in part C. Target is green with spheres for the I (cyan) and Q (green) residues of the IQ motif. CaM is gray except for CaMC helices F (red) and G (orange). Apo CaMbound to the NaV1.5 IQ motif (4OVN) or an IQ motif of myosin V (2IX7) shows I oriented into the semi-open cleft, and Q oriented towards the FG-linker in semi-open CaMC. Theintermediate state of CaM bound to the NaV1.2 IQ motif (4JPZ) retains the same orientation of CaMC as the apo state. Ca2+-saturated CaM adopts an open conformation bound to theIQ motif of NaV1.2 (2M5E) and myosin V (4ZLK) with Q oriented towards the junction between CaMN and CaMC. All 5 structures were aligned by residues corresponding to 1909–1915 of NaV1.2 in 2M5E, where I is 1912 and Q is at 1913. E. Alternative models of calcium-mediated response include (Ca2+)4-CaM dissociation from NaV1.2, which might promote(Ca2+)4-CaM binding at the DIII-DIV linker (“inactivation gate”, purple) as seen in 4DJC, or binding at a pair of CaMBD sequences (orange, cyan) that are not in a continuous helix,such as seen in 5SY1, the STRA6 receptor for retinol uptake. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

14 L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

that calcium triggers an ~180° rotation of CaMC around I1912 in the IQmotif, causing many changes at the binding interface between CaMC

and residues in the IQ motif. The final conformation is similar to thatof CaMC in (Ca2+)4-CaM bound to peptides from a variety of targetssuch as CaV2.2 (3DVJ [27]), CaMKI (1MXE [93]), and NaV1.5 III-IV linker(4DJC [40]).

To infer the biological occupancy of an IQ motif by one of theendstates of CaM (with 0 or 4 calcium ions bound), it is essential toknow both the equilibrium constants for binding the IQ motif and thelocal cellular concentration of free CaM. In cells, the level of CaMwill de-pend on the expression levels of target proteins and their post-transla-tional modifications, aswell as the affinity of CaM for each target [32,83,94–97]. The affinity is also a function of the degree of calciumoccupancyof CaM. To infer binding occupancy of theNaV1.2 IQmotif, we estimatedfree [CaM] as 50–100 nM [97,98]. Fig. 7A shows that this would corre-spond to 94–97% occupancy by apo CaM, and 37–54% occupancy by(Ca2+)4-CaM. This predicts that, regardless of the level of free calcium,CaMwould be constitutively bound to the NaV1.2 IQ motif. This predic-tion agrees with proteomics studies of NaV1.2-associated proteins inneuronal cells, which found the level of enrichment of CaM in NaV1.2pull-downs to be similar to that of other constitutively interacting pro-teins such as the NaV β2 subunit and FGF12 (FHF1) [11].

4.3. States of CaM-IQ: 0, 2, or 4 Ca2+

A cell experiences increases in calcium concentration due to releaseof calcium from internal stores or influx from the extracellular space. Acalcium titration in a cuvette or NMR tube mimics this transition. Titra-tions of CaM-IQpmonitored byNMR showed that calciumbound to CaMwith a stoichiometry of 4 ions to 1 CaM [16]. However, the calcium-binding affinity of sites III and IV (CaMC) was diminished, while the af-finity of sites I and II (CaMN) was similar to those sites in free CaM [16].Thus, the CaM domains behave differently and may bind calcium se-quentially but the CaM-IQp complex can attain full saturationwith 4 cal-cium ions bound.

Crystallographic structures of an intermediate state having half-sat-urated CaM bound to the IQ motifs of NaV1.2 (4JPZ) or NaV1.5 (4JQ0)provide insight into the transition during a calcium influx. Both struc-tures have calcium bound to sites I and II in CaMN, while sites III andIV are not saturated. The 4-helix bundles of CaMC in 4JPZ and 4JQ0 retainthe semi-open tertiary conformation and orientation characteristic ofapo CaM bound to the IQ motifs of NaV1.2 (2KXW) and NaV1.5 (2L53,4DCK, and 4OVN) shown in Fig. 2. These are similar to the groundbreak-ing structure (1G4Y, Fig. 2E) of CaM bound to a large fragment of the SKchannel [20] in which CaMC was apo, while CaMN was calcium-saturated.

The full ensemble of populated states of NaV1.2 changes in responseto the abundance of both CaM and calcium. In this study, we did not di-rectly measure calcium binding to the complex containing CaM boundto the NaV1.2 IQ motif. However, we can simulate this behavior basedon the difference in free energy (∆∆G) of binding apo and calcium-sat-urated CaM to the NaV1.2 IQ motif (1.93 kcal/mol in Table 1). Applyingthe principle of thermodynamic linkage, this value of ∆∆G must be thesame as the difference between the ∆G of calcium binding to free CaMand to the apo CaM-NaV1.2 complex. The simulation in Fig. 7B repre-sents predicted occupancy of the NaV1.2 IQ motif by (Ca2+)4-CaM.

Because the Kd for (Ca2+)4-CaM binding to the IQ motif is ~30-foldhigher (weaker) than that of apo CaM, the preferred binding partnerfor NaV1.2 at resting calcium conditions is apo CaM, which is depictedschematically in the blue vertical bar. The green bar signifies high calci-um; at 10 μM free calcium, occupancy of the IQ motif by (Ca2+)4-CaMwould be ~68% or two-thirds.While 10 μM free calciumdoes not persistin cells for long periods of time, and does not occur uniformly through-out the cell, the level of calciummay rise to this level transiently and lo-cally in nanodomains [99].

4.4. Rotation in place

A schematic diagram depicting the transition from 0 to 2 to 4 calci-um ions bound to CaM when associated with the NaV1.2 IQ motif isshown in Fig. 7C. In this model, calcium binding to sites I and II affectsonly CaMN, while calcium binding to sites III and IV triggers reversal ofCaMC orientation on the IQ motif. Our NMR data for apo and calcium-saturated CaM bound to Nav1.2IQp showed that backbone resonancesfor residues in CaMN in the CaM-IQP complex and free CaMN were es-sentially identical (Supp. Fig. S2) indicating that CaMN does not bindto NaV1.2IQP. This independence of CaMN was also seen in the NMRstructure (2L53, Fig. 1E) of apo CaM bound to NaV1.5. Thus, in Fig. 7C,CaMN is represented as a free domain, without a preferred interfaceon NaV1.2.

However, apo CaMN might adopt positions similar to the two dis-tinct, and well-separated locations seen in the crystallographic struc-tures for apo CaMN bound to NaV1.5 (4OVN or 4DCK, Fig. 1E), whilecalcium-saturated CaMN may adopt a position similar to those seen instructures 4JPZ or 4JQ0. The reversal of CaMC occurs with or withoutCaMN. Future studies in solution of CaM binding to a longer fragmentof NaV1.2 will be required to address to what extent the positions ofCaMN observed in crystallographic studies represent energetically pre-ferred orientations that are persistent in cells.

This simple 3-statemodel provides CaMwith a vocabulary of at leastthree words for allosteric regulation of NaV1.2. The larger question ishow thesemight alignwith three functional states of the channel broad-ly defined as closed, open and inactivated. However, each of those func-tional states is much more complex than a single protein conformation,and the physiological complexes include the NaV β subunits (with a sin-gle transmembrane helix), as well as auxiliary intracellular proteinssuch as intracellular FGFs (FHFs), ankyrins, CaMKII, synaptotagmin,and casein kinase II β which are also thought to modulate the NaV αsubunit [100,101]. Although our data cannot directly address the corre-lation between energetically accessible structures and physiologicalregulation of NaV1.2, they provide a striking new insight into an unex-pected structural response to calcium binding, and suggest that it willbe worth determining to what degree the calcium-saturated state ob-served in 2M5E is populated in cells.

4.5. Structural correlates of 3-state switch

CaM-target structures representing the 3 states depicted schemati-cally in Fig. 7C are shown in Fig. 7D. They were aligned using PyMOL(Schrödinger, LLC) according to the positions of residues correspondingto residues 1909–1915 of NaV1.2 in 2M5E, where I1912 (cyan) and Q1913

(green) in NaV1.2 are shown as ball-and-stick, and residues at positionscorresponding to those IQ residues are represented the sameway in theother structures. To highlight the orientation of CaM, CaMC helices E andH are gray, while F (red) and G (orange) draw attention to the reversalof CaMC relative to I1912 and Q1913. Apo CaM bound to the NaV1.5 IQmotif (4OVN) and to an IQmotif ofmyosin V (2IX7) show that the epon-ymous Q side chain is oriented towards the FG-linker in semi-open apoCaMC as it is in 2KXW. The intermediate state of CaM (having two calci-um ions bound to CaMN) associated with the NaV1.2 IQ motif (4JPZ) re-tains the same orientation of CaMC relative to the IQ motif as the apoform of CaM.

A recent report [102] of a calcium-triggered reversal of CaMC on IQmotif 1 ofmyosin V (4ZLK), shown in the right panel of Fig. 7D, is similarin orientation to 2M5E. In both structures, calciumbinding to CaMC trig-gers opening and reversal of the 4-helix bundle, with the side chain of Qoriented towards the linker between CaMN and CaMC. The observed po-larity of Nav1.2IQp relative to CaMC is consistent with most other com-plexes of calcium-saturated CaM bound to peptides [32] representingCaMBDs from other ion channels, receptors and enzymes such asCaMKII (1CDM) shown in Fig. 2A (termini of the CaMBD peptide(green) are indicated in blue/N and red/C).

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4.6. Reversal by release and recapture

How CaM could regulate NaV channels while remaining very tightlybound to “one” site within the C-terminal tail has been a long-standingconundrum. The thermodynamic and structural studies presented heredemonstrate that calciumbinding reverses the polarity of CaMbound tothe NaV1.2 IQ motif because CaMC recognizes nested, anti-parallel siteswithin a single helix in a calcium-dependent manner. This unexpectedre-orientation of CaM illustrates the exquisite power of calcium-in-duced helical reorientations to result in large re-orientation of CaM toyield regulatory effects. Although studies here only addressed interac-tions of CaMwith the IQmotif of NaV1.2, they suggest several hypothe-ses about the regulatory action of CaM on the channel as a whole.Because calcium binding to CaMC triggers it to rotate in the saddle ofthe hydrophobic face of the IQ motif, this might assist in modulatingNaV1.2 by moving CaMN into an alternative position that could reachother sequences within NaV1.2 or its auxiliary regulatory proteins.Based on studies of CaM-NaV interactions, Young and Caldwell postu-lated that NaV1.4 might contain an NLBD – a non-IQ domain thatbinds CaMN [101].

The observed calcium-induced reversal of CaM also provides a plau-sible mechanism for transient release of CaM from the NaV1.2 IQ motif,despite its very high affinity for both apo and calcium-saturated CaM.This would be analogous to IQCG (IQ motif-containing G protein) inwhich calcium causes release of CaMC but promotes binding of(Ca2+)2-CaMN [103]. Although none of the experiments reported heredirectly address the kinetic aspects of binding or release of CaM fromthe channel or the IQ motif, Fig. 7E illustrates some plausible modelsbased on known CaM-target interactions. One study concluded that dis-sociated (Ca2+)4-CaM acts on NaV1.5 indirectly by binding to CaMKIIwhich then phosphorylates the channel [101]. Other studies of NaV1.5have proposed that high levels of calcium may cause CaM to releasefrom the IQ motif and relocate at least one of its domains to the DIII-DIV linker, which has micromolar affinity for CaMC [39,41]. For NaV1.5,(Ca2+)4-CaM binds at the DIII-DIV linker (“inactivation gate”) throughCaMC as shown in the crystallographic structure 4DJC, which has thesame polarity of CaMC-peptide interaction observed in many other cal-cium-saturated CaM complexes such as 1CDM (Fig. 2A). Given that thestructure of the CTD of NaV1.5 (4OVN, 4DCK, 4JQ0) is highly similar tothe CTD of NaV1.2 (4JPZ), release of (Ca2+)4-CaM from the IQ motifmight result in one or both CaM domains binding the NaV1.2 DIII-DIVlinker, post-IQ or other non-IQ CaM-binding siteswithin the CTD. Prece-dents exist for CaM binding the N-terminal tail of CaV channels [44,45];NaVs may have a similar site. (Ca2+)4-CaMmay bind to the NaV β sub-units or other auxiliary proteins such as those depicted in models ofNaV1.5 [104].

Re-association or relocation of (Ca2+)4-CaM might occur as it doesfor the STRA6 receptor for retinol uptake in which the two domains ofCaM bind and bridge distinct sequences in the receptor that are notfound in a continuous helix [105]. This was also observed for CaM bind-ing to non-inactivating voltage-dependent KCNQ potassium channels[106]. Another plausible arrangement might follow the example ofCaMbinding to the SK Channel (1G4Y [20]) wherein a second CaMmol-eculemay be recruited toNaV1.2when calcium levels rise. Such amech-anism would share features with the calcium-induced remodeling ofmyosin V, where one CaM molecule has been observed to bind two IQmotifs simultaneously [85].

4.7. CaM-regulated channels across phyla

By studying Paramecium — a “unicellular neuron” — Kung and col-leaguesfirst discovered that CaMwas a constitutive subunit of some cal-cium-regulated sodium and potassium channels, and that defects ineach domain of CaM compromise a unique set of physiological re-sponses to external stimuli [107–110]. Under-reactive mutants of thisexcitable cell deviated from wild-type CaM at positions in CaMN that

were primarily outside of the calcium-binding sites, while over-reactivemutants had changes in CaMC that were primarily localized within sitesIII and IV. Similar domain-specific results were later observed in muta-genic studies in yeast [111]. Exome analyses of humans have identifiedcalmodulinopathies that are also specific to one domain of CaM [112–115]. Some of the identified positions, such as D95 in site III, havebeen found to be deleterious in both Paramecium and human studies,suggesting that their mechanisms of action are conserved.

Given that the sequences of Paramecium and human CaM are 89%identical, and 96% similar, and that all of the mutants identified byKungwere at positions that are identical in the human and Parameciumwild-type CaM sequences, it was predictable that the initial discovery ofCaM-regulated ion channels would generalize to several families of cal-cium-regulated ion channels in all eukaryotes [92,116–119]. For manyof these channels, CaM is associated under resting (low calcium) condi-tions as it is with NaV1.2. CaMmay activate, inhibit, or do both, depend-ing on its fractional saturation by calcium. For example, studies of theryanodine receptor RyR1 [120–122] showed that apo CaM activated it,while calcium-saturated CaM inhibited it, showing that switchingcould be promoted by the addition or dissociation of calcium.

4.8. Power of protein ensembles

Prior to its recognition as a regulator of ion channels, studies of CaMrevolved around its regulation of soluble enzymes including kinases, cy-clases, phosphodiesterases, and phosphatases which are activated by(Ca2+)4-CaM [123–126]. The two domains or lobes of CaMwere viewedprimarily as resulting from duplication of the gene for a primordial 4-helix bundle with metal-binding loops [127–130]. Having two sets ofpaired EF-hands could be an evolutionary advantage for increasingavidity for targets in signaling pathways and enlarging the recognitionsite, thereby amplifying the response to an influx of calcium from theexternal medium, or internal stores. As membrane-spanning receptorsand channels came to be recognized as non-enzymatic targets of CaM,some of them, such as NMDA [79,131,132], RyR1 [133–135] andCaV1.2 [91], were found to bind apo CaM under resting conditions, buthave a higher affinity for calcium-saturated CaM than apo CaM. In thatrespect, they were similar to kinases such as CaMKII.

Clearly, calcium-saturated CaM is a biologically significant state ofCaM. In yeast, Davis and colleagues also demonstrated that apo CaM isessential to viability [136–138]. There are 16 states total: 14 possible in-termediates with 1, 2 or 3 calcium ions bound. Structures of (Ca2+)2-CaMbound to a fragment of the SK channel (1G4Y), and the IQmotif re-gion of NaV1.2 (4JPZ) and NaV1.5 (4JQ0) are examples of an intermedi-ate partially ligated CaM. Half-saturated CaM has also been observed inreverse –with apo CaMN and calcium-saturated CaMC bound to the an-thrax edema factor [139]. It is even possible that CaMwith one or threecalcium ions bound may also have a significant role in some physiolog-ical processes. CaBP1 (Calcium Binding Protein 1) binds only 3 Ca2+,suggesting that there are some neuronal targets that prefer this stoichi-ometry [140].Wild-type CaMwith 4 functional sites is unlikely to adoptstates with only one site filled in a domain because of the high degree ofcooperativity between calcium-binding sites in the same domains.However, interactions between binding-site residues in CaM and sidechains of residues in a target protein could alter the energetic balanceto tip it in favor of an unoccupied site as proposed in the analysis of4DCK [22].

In neurons, sodium channelswithmultiple binding sites for auxiliaryproteins integrate input from a panoply ofmodulators including CaM. Inthe structure reported here (2M5E), we showed that calcium saturationof CaM bound to the IQ motif of NaV1.2 triggers a half-turn rotation ofCaMC around I1912 which has structural consequences for the geometryof CaMN as well as CaMC. The physiological consequences must be ex-plored further. Having determined what CaM does on an isolated IQmotif, it remains to be seen whether CaM reverses polarity in solutionon the full CTD of NaV1.2, and how the binding of intracellular FGFs

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(FHFs) may influence CaM-IQ interactions. Just as regulatory pathwaysin genetic circuits quantitatively integrate the effects of multiple pro-teins binding to DNA, and interacting both directly and allostericallywith each other [141], neurons can take advantage of the full ensembleof energetically accessible states for auxiliary proteins binding tochannels, and make use of ligand-triggered changes in their flexiblestructures.

4.9. Accession numbers

The coordinates for the average minimized structure and 20 lowestenergy models were deposited with the PDB code of 2M5E; therewere preliminary conference reports of this structure [142,143]. NMRassignment data (accession number 19050) were deposited with theBioMagResBank (BMRB http://www.bmrb.wisc.edu). Assignments arealso contained in Supplemental Tables S2 and S3.

Acknowledgments

We thank A. Persechini and DJ Black for sharing a published vectorused as the parent for the biosensor overexpression plasmidmade in thisstudy, and Sean A. Klein for critical input on experimental approaches.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bpc.2017.02.006.

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19L. Hovey et al. / Biophysical Chemistry 224 (2017) 1–19

Filename:SuppTable1A_HoveyEtAl_2M5E_NaV1.2_IQ_53seqs.xlsx

Hovey et al, Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in theIQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2

Supplementary Table S1ASupports Figure 1 - WebLogo

IQSequenceTable(CompletelyConservedPositionsHighlightedinYellow)R K E A I Q R

Species CommonName StartResidue 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 EndResidue IQ Sequence Text1 Homosapiens Human 1901 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIIIQRAYRRYLLKQKVKK2 Ailuropodamelanoleuca GiantPanda 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK3 Anasplatyrhynchos Mallard 1903 K R K Q E E V S A V V I Q R A F R R H L L R Q K V K K 1929 KRKQEEVSAVVIQRAFRRHLLRQKVKK4 Anoliscarolinensis Carolinaanole 1859 K R K Q E E V S A V I I Q R A F R S Y L L R Q K V K K 1885 KRKQEEVSAVIIQRAFRSYLLRQKVKK5 Bostaurus Cow 1902 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1928 KRKQEEVSAIVIQRAYRRYLLKQKVKK6 Callithrixjacchus Commonmarmoset 1901 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIIIQRAYRRYLLKQKVKK7 Canislupusfamiliaris Dog 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK8 Caviaporcellus Guineapig 1865 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1891 KRKQEEVSAIVIQRAYRRYLLKQKVKK9 Ceratotheriumsimumsimum Whiterhinoceros 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK10 Cheloniamydas Greenseaturtle 1900 K R K Q E E V S A T V I Q R A Y R C Y I L K Q K V K K 1926 KRKQEEVSATVIQRAYRCYILKQKVKK11 Condyluracristata Star-nosedmole 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK12 Cricetulusgriseus Chinesehamster 1853 K R K Q E E V S A I V I Q R A Y R R Y V L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYVLKQKVKK13 Dasypusnovemcinctus Nine-bandedarmadillo 1901 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIVIQRAYRRYLLKQKVKK14 Echinopstelfairi Lesserhedgehogtenrec 1839 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K I K K 1865 KRKQEEVSAIVIQRAYRRYLLKQKIKK15 Equuscaballus Horse 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK16 Feliscatus Cat 1853 K R K Q E E V S A V V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAVVIQRAYRRYLLKQKVKK17 Ficedulaalbicollis CollaredFlycatcher 1903 K R K Q E E V S A V I I Q R A Y R R H L L R L K V K K 1929 KRKQEEVSAVIIQRAYRRHLLRLKVKK18 Gallusgallus RedJunglefowl 1908 K R K Q E E V S A L I I Q R A Y R C Y L L K Q K V K K 1934 KRKQEEVSALIIQRAYRCYLLKQKVKK19 Gorillagorillagorilla Westernlowlandgorilla 1910 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1936 KRKQEEVSAIIIQRAYRRYLLKQKVKK20 Heterocephalusglaber Nakedmolerat 1902 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1928 KRKQEEVSAIVIQRAYRRYLLKQKVKK21 Jaculusjaculus LesserEgyptianjerboa 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK22 Loxodontaafricana Africanbushelephant 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK23 Macacamulatta Rhesusmacaque 1901 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIIIQRAYRRYLLKQKVKK24 MaylandiaZebra ZebraMbuna 1611 R R K Q E E M S A I I I Q R A F R R Y M I R L A M K K 1637 RRKQEEMSAIIIQRAFRRYMIRLAMKK25 Meleagrisgallopavo WildTurkey 1248 K R K Q E E V S A V V I Q R A F R R H L L R Q K V K K 1274 KRKQEEVSAVVIQRAFRRHLLRQKVKK26 Melopsittacusundulatus Budgerigar 1888 K R K Q E E V S A V I I Q R A Y R R H L L R Q K V K K 1914 KRKQEEVSAVIIQRAYRRHLLRQKVKK27 Mesocricetusauratus Goldenhamster 1722 K R K Q E E V S A I V I Q R A Y R R Y V L K Q K V K K 1748 KRKQEEVSAIVIQRAYRRYVLKQKVKK28 Monodelphisdomestica Grayshort-tailedopossum 1853 K R K Q E E V S A I I I Q R A Y R R H L L R Q K V K K 1879 KRKQEEVSAIIIQRAYRRHLLRQKVKK29 Musmusculus Housemouse 1902 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1928 KRKQEEVSAIVIQRAYRRYLLKQKVKK30 Mustelaputoriusfuro Ferret 1902 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1928 KRKQEEVSAIVIQRAYRRYLLKQKVKK31 Myotisdavidii Mouse-earedbat 1710 K R K Q E E V S A T I I Q R A Y R C Y L L R Q K V K K 1736 KRKQEEVSATIIQRAYRCYLLRQKVKK32 Nomascusleucogenys Northernwhite-cheekedgibbon 1901 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIIIQRAYRRYLLKQKVKK33 Ochotonaprinceps Americanpika 1821 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1847 KRKQEEVSAIVIQRAYRRYLLKQKVKK34 Octodondegus Degu 1782 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1808 KRKQEEVSAIVIQRAYRRYLLKQKVKK35 Odobenusrosmarusdivergens Walrus 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK36 Orcinusorca Killerwhale 1810 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1836 KRKQEEVSAIVIQRAYRRYLLKQKVKK37 Ornithorhynchusanatinus Platypus 1853 K R K Q E E V S A I I I Q R A Y R R Y L L R Q K V K K 1879 KRKQEEVSAIIIQRAYRRYLLRQKVKK38 Oryctolaguscuniculus Europeanrabbit 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK39 Ovisaries Sheep 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK40 Panpaniscus Bonobo 1901 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIIIQRAYRRYLLKQKVKK41 Pantroglodytes Commonchimpanzee 1901 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIIIQRAYRRYLLKQKVKK42 Papioanubis OliveBaboon 1821 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1847 KRKQEEVSAIIIQRAYRRYLLKQKVKK43 Pteropusalecto Blackflyingfox 1857 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1883 KRKQEEVSAIVIQRAYRRYLLKQKVKK44 Rattusnorvegicus Brownrat 1901 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIVIQRAYRRYLLKQKVKK45 Saimiriboliviensisboliviensis Black-cappedsquirrelmonkey 1901 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1927 KRKQEEVSAIIIQRAYRRYLLKQKVKK46 Sarcophilusharrisii Tasmaniandevil 1903 K R K Q E E V S A V I I Q R A Y R R H L L R Q K V K K 1929 KRKQEEVSAVIIQRAYRRHLLRQKVKK47 Sorexaraneus Commonshrew 1903 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1929 KRKQEEVSAIVIQRAYRRYLLKQKVKK48 Susscrofa Wildboar 1853 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1879 KRKQEEVSAIVIQRAYRRYLLKQKVKK49 Taeniopygiaguttata ZebraFinch 1903 K R K Q E E V S A V I I Q R A Y R R H L L R Q K V K K 1929 KRKQEEVSAVIIQRAYRRHLLRQKVKK50 Takifugurubripes Takifugupufferfish 1857 R R K L E D T S A T V I Q R A Y R Q Y A S Q R S P V K 1883 RRKLEDTSATVIQRAYRQYASQRSPVK51 Trichechusmanatuslatirostris WestIndianmanatee 1810 K R K Q E E V S A I V I Q R A Y R R Y L L K Q K V K K 1836 KRKQEEVSAIVIQRAYRRYLLKQKVKK52 Tupaiachinensis Tupaia 1523 K R K Q E E V S A I I I Q R A Y R R Y L L K Q K V K K 1549 KRKQEEVSAIIIQRAYRRYLLKQKVKK53 Xenopus(Silurana)tropicalis Westernclawedfrog 1904 R R K Q E E L A A I V I Q R C Y R C Y A L R K G I K H 1930 RRKQEELAAIVIQRCYRCYALRKGIKH

TableSupportingFigure1of Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2 Liam Hovey1,+, C. Andrew Fowler2,+, Ryan Mahling1, Zesen Lin1, Mark Stephen Miller1, Dagan C. Marx1, Jesse B. Yoder1, Elaine H. Kim1, Kristin M. Tefft1, Brett C. Waite1, Michael D. Feldkamp1, Liping Yu2, Madeline A. Shea1,*1 Department of Biochemistry, and 2 NMR Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109Corresponding Author: Madeline A. Shea, madeline-shea@uiowa.edu, (319) 335-7885

Comparison of NaV1.2 from 53 species

Numbering based on Human NaV1.2WebLogo 3.3

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RKRKLQ

1905

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1915

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I

VALS

I

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K

L

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G

A

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P

M

IVV

KH

K

Filename:SuppTable1B_HoveyEtAl_2M5E_NaV1.5_IQ_40seqs.xlsx

Hovey et al, Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in theIQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.5

Supplementary Table S1BSupports Figure 1 - WebLogo

IQSequenceTable(CompletelyConservedPositionsHighlightedinYellow)R K E E V S I Q R A R H L R

Species CommonName StartResidue 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 EndResidue IQ Sequence Text1 Homosapiens Human 1897 R R K H E E V S A M V I Q R A F R R H L L Q R S L K H 1918 RRKHEEVSAMVIQRAFRRHLLQRSLKH2 Ailuropodamelanoleuca Giantpanda 1890 R R K H E E V S A T I I Q R A F R R H L L Q R S M K H 1916 RRKHEEVSATIIQRAFRRHLLQRSMKH3 Anoliscarolinensis Greenanole 1575 R R K H E E V S A I L I Q R A Y R S H L F Q R S L K Q 1601 RRKHEEVSAILIQRAYRSHLFQRSLKQ4 Aotusnancymaae NancyMa'snightmonkey 1908 R R K Q E E V S A T V I Q R A F R R H L L Q R S L K H 1934 RRKQEEVSATVIQRAFRRHLLQRSLKH5 Bostaurus Bovine 1902 R R K H E E V S A T I I Q R A F R R H L L Q R S V K H 1928 RRKHEEVSATIIQRAFRRHLLQRSVKH6 Callithrixjacchus White-tufted-earmarmoset 1843 R R K H E E V S A T V I Q R A F R R H L L Q R S M K H 1869 RRKHEEVSATVIQRAFRRHLLQRSMKH7 Canislupusfamiliaris Dog 1760 R R K H E E V S A T V I Q R A F R R H L L Q R S M K H 1786 RRKHEEVSATVIQRAFRRHLLQRSVKH8 Caviaporcellus Gunieapig 1895 R R K H E E V S A T V I Q R A F R R H L L Q R S M K H 1912 RRKHEEVSATVIQRAFRRHLLQRSMKH9 Cercocebusatys Sootymangabey 1645 R R K H E E V S A M V I Q R A F R R H L L Q R S V K H 1617 RRKHEEVSAMVIQRAFRRHLLQRSVKH10 Chlorocebussabaeus Greenmonkey 1762 R R K H E E V S A M V I Q R A F R R H L L Q R S V K H 1788 RRKHEEVSAMVIQRAFRRHLLQRSVKH11 Colobusangolensispalliatus OldWorldmonkey 1736 R R K H E E V S A M V I Q R A F R R H L L Q R S V K H 1762 RRKHEEVSAMVIQRAFRRHLLQRSVKH12 Equuscaballus Horse 1845 R R K H E E V S A T I I Q R A F R R H L L Q R S M K H 1871 RRKHEEVSATIIQRAFRRHLLQRSMKH13 Feliscatus Cat 1899 R R K H E E V S A T V I Q R A F R R H L L Q R S M K H 1925 RRKHEEVSATVIQRAFRRHLLQRSMKH14 Ficedulaalbicollis Collaredflycatcher 1922 R R K Q E E V S A I V I Q R A Y R R H L F R R S M K Q 1948 RRKQEEVSAIVIQRAYRRHLFRRSMKQ15 Galeopterusvariegatus Sundaflyinglemur 1845 R R K H E E V S A T V I Q R A F R R H L L Q R S V K H 1871 RRKHEEVSATVIQRAFRRHLLQRSVKH16 Gorillagorillagorilla Westernlowlandgorilla 1843 R R K H E E V S A T V I Q R A F R R H L L Q R S L K H 1869 RRKHEEVSATVIQRAFRRHLLQRSLKH17 Ictaluruspunctatus Channelcatfish 1834 R R K Q E E V S S I V I Q R A Y R R H L L R R Q L R Q 1860 RRKQEEVSSIVIQRAYRRHLLRRQLRQ18 Macacafascicularis Crab-eatingmacaque 1897 R R K H E E V S A M V I Q R A F R R H L L Q R S V K H 1923 RRKHEEVSAMVIQRAFRRHLLQRSVKH19 Macacamulatta Rhesusmacaque 1691 R R K H E E V S A M V I Q R A F R R H L L Q R S V K H 1717 RRKHEEVSAMVIQRAFRRHLLQRSVKH20 Macacanemestrina Southernpig-tailedmacaque 1897 R R K H E E V S A M V I Q R A F R R H L L Q R S V K H 1923 RRKHEEVSAMVIQRAFRRHLLQRSVKH21 Mandrillusleucophaeus Drill 1842 R R K H E E V S A M V I Q R A F R R H L L Q R S V K H 1868 RRKHEEVSAMVIQRAFRRHLLQRSVKH22 Meleagrisgallopavo Commonturkey 1915 R R K Q E E V S A I V I Q R A Y R R H L F R R S M K Q 1941 RRKQEEVSAIVIQRAYRRHLFRRSMKQ23 Mesocricetusauratus Goldenhamster 1902 R R K H E E V S A T V I Q R A F R R H L L Q R S V K H 1928 RRKHEEVSATVIQRAFRRHLLQRSVKH24 Monodelphisdomestica Grayshort-tailedopossum 1722 R R K H E E V S A T V I Q R A F R R H L L Q R S V K H 1748 RRKHEEVSATVIQRAFRRHLLQRSVKH25 Musmusculus Mouse 1900 R R K H E E V S A T V I Q R A F R R H L L Q R S V K H 1926 RRKHEEVSATVIQRAFRRHLLQRSVKH26 Mustelaputoriusfuro Europeandomesticferret 1896 R R K H E E V S A T I I Q R A F R R H L L Q R S M K H 1922 RRKHEEVSATIIQRAFRRHLLQRSMKH27 Myotisbrandtii Brandt'sbat 427 R R K H E E V S A T I I Q R A F R R H L L Q R S M K H 502 RRKHEEVSATIIQRAFRRHLLQRSMKH28 Myotislucifugus Littlebrownbat 1770 R R K H E E V S A T I I Q R A F R R H L L Q R S M K Q 1796 RRKHEEVSATIIQRAFRRHLLQRSMKQ29 Nomascusleucogenys Nothernwhite-cheekedgibbon 1895 R R K H E E V S A T V I Q R A F R R H L L Q R S L K H 1921 RRKHEEVSATVIQRAFRRHLLQRSLKH30 Ornithorhynchusanatinus Duckbillplatypus 1169 R R K H E E V S A I V I Q R A F R S H L L R R S V K H 1195 RRKHEEVSAIVIQRAFRSHLLRRSVKH31 Otolemurgarnettii small-earedgalago 1900 R R K H E E V S A T V I Q R A F R R H L L Q R S M K H 1926 RRKHEEVSATVIQRAFRRHLLQRSMKH32 Ovisaries sheep 1812 S R K H E E V S A T V I Q R A S R R H L L R R S V K P 1838 SRKHEEVSATVIQRASRRHLLRRSVKP33 Panpaniscus Bonobo 1896 R R K H E E V S A T V I Q R A F R R H L L Q R S L K H 1922 RRKHEEVSATVIQRAFRRHLLQRSLKH34 Pantroglodytes Commonchimpanzee 1897 R R K H E E V S A T V I Q R A F R R H L L Q R S L K H 1923 RRKHEEVSATVIQRAFRRHLLQRSLKH35 Peromyscusmaniculatusbairdii Deermouse 1860 R R K H E E V S A T V I Q R A F R R H L L Q R S V K H 1886 RRKHEEVSATVIQRAFRRHLLQRSVKH36 Rattusnorvegicus Rat 1843 R R K H E E V S A T V I Q R A F R R H L L Q R S V K H 1869 RRKHEEVSATVIQRAFRRHLLQRSVKH37 Rhinopithecusroxellana Goldensnub-nosedmonkey 1763 R R K H E E V S A M V I Q R A F R R H L L Q R S V K H 1789 RRKHEEVSAMVIQRAFRRHLLQRSVKH38 Saimiriboliviensisboliviensis Black-cappedsquirrelmonkey 1896 R R K H E E V S A T V I Q R A F R R H L L Q R S M K H 1922 RRKHEEVSATVIQRAFRRHLLQRSMKH39 Susscrofa pig 1901 R R K H E E V S A T I I Q R A F R R H L L Q R S V K H 1927 RRKHEEVSATIIQRAFRRHLLQRSVKH40 Xenopustropicalis Westernclawedfrog 1864 K R K Q E E V S A C V I Q R A Y R R H L L C R S M K Q 1890 KRKQEEVSACVIQRAYRRHLLCRSMKQ

TableSupportingFigure1of Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2 Liam Hovey1,+, C. Andrew Fowler2,+, Ryan Mahling1, Zesen Lin1, Mark Stephen Miller1, Dagan C. Marx1, Jesse B. Yoder1, Elaine H. Kim1, Kristin M. Tefft1, Brett C. Waite1, Michael D. Feldkamp1, Liping Yu2, Madeline A. Shea1,*1 Department of Biochemistry, and 2 NMR Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109Corresponding Author: Madeline A. Shea, madeline-shea@uiowa.edu, (319) 335-7885

Comparison of NaV1.5 from 40 species

Numbering based on Human NaV1.5

0.0

1.0

2.0

3.0

4.0

bits

SKRRK

1900

QHEEVS

1905

SAC

IMTL

IVIQ

1910

RASYFRSR1915

HLFLCRQR1920

Q

SLMVRKPQH

Filename:SuppTable1C_HoveyEtAl_2M5E_NaV1.6_IQ_45seqs.xlsx

Hovey et al, Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in theIQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.6

Supplementary Table S1CSupports Figure 1 - WebLogo

IQSequenceTable(CompletelyConservedPositionsHighlightedinYellow)R R K Q E S A A R L G

Species CommonName StartResidue 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 EndResidue IQ Sequence Text1 Homosapiens Human 1889 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1915 RRKQEEVSAVVLQRAYRGHLARRGFIC2 Ailuropodamelanoleuca Giantpenda 1827 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1853 RRKQEEVSAVVLQRAYRGHLARRGFIC3 Anoliscarolinensis Greenanole 1885 R R K Q E E V S A L V I Q R A Y R A R L V R R G F Y R 1910 RRKQEEVSALVIQRAYRARLVRRGFYR4 Bostaurus Bovine 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC5 Bubalusbubalis waterbuffalo 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC6 Callithrixjacchus Commonmarmoset 1849 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1875 RRKQEEVSAVVLQRAYRGHLARRGFIC7 Camelusferus Wildbacteriancamel 928 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 954 RRKQEEVSAVVLQRAYRGHLARRGFIC8 Canislupusfamiliaris Dog 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC9 Ceratotheriumsimumsimum Whiterhinoceros 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC10 Cercocebusatys Sootymangabey 1902 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1928 RRKQEEVSAVVLQRAYRGHLARRGFIC11 Chrysochlorisasiatica Capegoldenmole 1890 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1916 RRKQEEVSAVVLQRAYRGHLARRGFIC12 Daniorerio Zebrafish 1860 R R K Q E H M S A G V I Q R A F R A H L I R K G F I C 1886 RRKQEHMSAGVIQRAFRAHLIRKGFIC13 Equusasinus Donkeys 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC14 Equuscaballus Horse 1890 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1916 RRKQEEVSAVVLQRAYRGHLARRGFIC15 Erinaceuseuropaeus Europeanhedgehog 1890 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1916 RRKQEEVSAVVLQRAYRGHLARRGFIC16 Feliscatus Cat 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F V C 1917 RRKQEEVSAVVLQRAYRGHLARRGFVC17 Ficedulaalbicollis Collaredflycatcher 1740 R R K Q E E V S A V L M M R A Y R A R L A R R G F V G 1766 RRKQEEVSAVLMMRAYRARLARRGFVG18 Galeopterusvariegatus Sundaflyinglemur 1740 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1765 RRKQEEVSAVVLQRAYRGHLARRGFIC19 Gallusgallus Chicken 1889 R R K Q E E V S A V V L Q R A Y R S R L A R R G G F W 1912 RRKQEEVSAVVLQRAYRSRLARRGGFW20 Loxodontaafriicana Africanelephant 1902 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1928 RRKQEEVSAVVLQRAYRGHLARRGFIC21 Macacamulatta Rhesusmacaque 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC22 Macacanemestrina Southernpig-tailedmacaque 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC23 Microcebusmurinus Graymouselemur 1901 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1927 RRKQEEVSAVVLQRAYRGHLARRGFIC24 Musmusculus Mouse 1848 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1874 RRKQEEVSAVVLQRAYRGHLARRGFIC25 Mustelaputoriusfuro Europeandomesticferret 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC26 Myotislucifugus Littlebrownbat 1888 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1914 RRKQEEVSAVVLQRAYRGHLARRGFIC27 Nomascusleucogenys Northernwhite-cheekedgibbon 1902 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1928 RRKQEEVSAVVLQRAYRGHLARRGFIC28 Orycteropusaferafer Aardvark 1715 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1741 RRKQEEVSAVVLQRAYRGHLARRGFIC29 Oryctolaguscuniculus Rabbit 1902 R R K Q E E V S A V V L Q R A Y R G H L A R R G F V C 1928 RRKQEEVSAVVLQRAYRGHLARRGFVC30 Ovisariesmusimon Mouflon 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC31 Panpaniscus Bonoboo 1902 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1928 RRKQEEVSAVVLQRAYRGHLARRGFIC32 Pantroglodytes Chimpanzee 1850 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1876 RRKQEEVSAVVLQRAYRGHLARRGFIC33 Papioanubis Olivebaboon 1890 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1916 RRKQEEVSAVVLQRAYRGHLARRGFIC34 Pelodiscussinensis Chinesesoftshellturtle 1891 R R K Q E D V S A V V I Q R A Y R V R L A K R G F I C 1917 RRKQEDVSAVVIQRAYRVRLAKRGFIC35 Pongoabelii Sumatranorangutan 1736 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1762 RRKQEEVSAVVLQRAYRGHLARRGFIC36 Pongopygmaeus Borneanorangutan 1754 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1780 RRKQEEVSAVVLQRAYRGHLARRGFIC37 Propithecuscoquereli Coquerel'ssifaka 1849 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1875 RRKQEEVSAVVLQRAYRGHLARRGFIC38 Pteropusalecto Blackflyingfox 411 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 437 RRKQEEVSAVVLQRAYRGHLARRGFIC39 Rattusnorvegicus Brownrat 1889 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1915 RRKQEEVSAVVLQRAYRGHLARRGFIC40 Sarcophilusharrisii Tasmaniandevil 1889 R R K Q E D V S A V V L Q R A Y R G H L A R R G F I S 1915 RRKQEDVSAVVLQRAYRGHLARRGFIS41 Taeniopygiaguttata Zebrafinch 1894 R R K Q E E V S A V V I Q R A Y R A R L A R R G F V G 1920 RRKQEEVSAVVIQRAYRARLARRGFVG42 Trichechusmanatuslatirostris WestIndianmanatee 1850 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1876 RRKQEEVSAVVLQRAYRGHLARRGFIC43 Tupaiachinensis Chinesetreeshrew 618 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 644 RRKQEEVSAVVLQRAYRGHLARRGFIC44 Vicugnapacos Alpaca 1891 R R K Q E E V S A V V L Q R A Y R G H L A R R G F I C 1917 RRKQEEVSAVVLQRAYRGHLARRGFIC45 Xenopustropicalis Westernclawedfrog 1872 R R K Q E D V S A V V I Q Q A Y R R H L V K R G F I S 1898 RRKQEDVSAVVIQQAYRRHLVKRGFIS

TableSupportingFigure1of Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2 Liam Hovey1,+, C. Andrew Fowler2,+, Ryan Mahling1, Zesen Lin1, Mark Stephen Miller1, Dagan C. Marx1, Jesse B. Yoder1, Elaine H. Kim1, Kristin M. Tefft1, Brett C. Waite1, Michael D. Feldkamp1, Liping Yu2, Madeline A. Shea1,*1 Department of Biochemistry, and 2 NMR Facility, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109Corresponding Author: Madeline A. Shea, madeline-shea@uiowa.edu, (319) 335-7885

Comparison of NaV1.6 from 45 species

Numbering based on Human NaV1.6

0.0

1.0

2.0

3.0

4.0

bits R

1890

RKQEHDE1895

MVSALGVLV

1900

M

ILM

QQRAFY

1905

RVSRAGRHLIVA1910

KRKRG

GFY

F

VI

1915

W

R

SG

C

Hovey et al, Calcium Triggers Reversal of Calmodulin on Nested Anti-Parallel Sites in the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2

Supplementary Table 2 Supports Table 2 - Structural Statistics for 2M5E.pdb

Chemical shifts of 13C,15N labeled, (Ca2+)2-PCaM76-148 bound to Nav1.2IQp. Red boxes indicate missing assignments. Blue boxes indicate missing methylene proton assignments that may be degenerate.

Residue HN N Nδ Nε Hα Hβ Hγ Hδ Hε Hζ C Cα Cβ Cγ Cδ Cε Cζ 76Met - - - - - - - - 2.10 - - - - - - 16.9 -

77Lys - - - - 4.35 1.82 1.44 1.71 3.00 - 176.2 56.4 32.9 24.5 28.9 41.9 -

78Glu 8.75 123.2 - - 4.23 1.96 2.29 - - - 176.5 57.1 30.0 36.3 - - -

79Gln 8.43 120.5 - 112.2 4.34 2.11

2.032.37

- 7.61

6.83- 175.7 56.0 29.6 33.8 - - -

80Asp 8.41 121.7 - - 4.65 2.712.66

- - - - 176.3 54.5 41.3 - - - -

81Ser 8.38 116.2 - - 4.50 4.073.96

- - - - 175.2 58.6 64.0 - - - -

82Glu 8.64 123.0 - - 4.22 2.08 2.35 - - - 177.4 58.6 29.7 36.4 - - -

83Glu 8.44 118.8 - - 4.07 2.10 2.36 - - - 178.5 59.4 29.5 36.5 - - -

84Glu 8.13 118.9 - - 4.23 2.332.21

2.432.32

- - - 178.9 59.0 30.0 36.9 - - -

85Leu 8.03 121.1 - - 4.32 2.341.66

1.83 0.890.82

- - 178.9 57.9 42.0 27.3 26.323.4

- -

86Ile 8.23 121.1 - - 3.82 1.88 1.810.880.86

0.64 - - 178.2 64.9 38.2 28.917.3

13.3 - -

87Glu 7.72 118.3 - - 4.01 2.16 2.37 - - - 179.2 59.1 29.4 36.5 - - -

88Ala 8.04 121.3 - - 3.88 1.13 - - - - 178.6 55.2 17.3 - - - -89Phe 8.44 118.9 - - 2.97 3.18

2.95- 6.63 7.05 6.92 176.9 62.3 39.2 - 131.7 131.3 131.8

90Lys 7.65 114.7 - - 3.83 1.88 1.881.57

1.69 2.91 - 178.1 59.0 32.8 25.9 29.7 41.8 -

91Val 7.29 118.7 - - 3.29 2.03 0.780.11

- - - 176.5 65.6 31.0 23.120.2

- - -

92Phe 6.79 115.1 - - 4.13 2.652.43

- 7.25 7.09 7.22 175.7 60.6 40.7 - 131.8 130.5 129.2

93Asp 7.72 115.8 - - 4.51 2.221.34

- - - - 177.6 51.9 38.5 - - - -

94Arg 7.51 124.1 - - 3.82 1.901.74

1.741.59

3.09 - - 178.0 59.5 30.9 27.0 43.2 - -

95Asp 8.28 113.8 - - 4.53 3.072.63

- - - - 178.0 53.0 39.7 - - - -

96Gly 7.74 108.7 - - 3.863.86

- - - - - 175.0 47.1 - - - - -

97Asn 8.20 119.4 115.3 - 4.61 3.272.60

- 7.966.82

- - 176.1 52.6 37.8 - - - -

98Gly 10.85 113.5 - - 4.273.64

- - - - - 173.0 45.3 - - - - -

99Leu 7.49 119.1 - - 4.89 1.570.96

1.33 0.880.44

- - 175.8 52.7 46.3 25.8 25.223.5

- -

100Ile 9.96 128.3 - - 4.71 1.96 1.340.950.12

0.40 - - 175.8 61.5 38.8 26.817.7

15.7 - -

101Ser 9.27 124.5 - - 4.78 4.384.00

- - - - 174.8 55.5 67.1 - - - -

102Ala 9.22 123.2 - - 3.87 1.42 - - - - 179.3 55.7 17.9 - - - -103Ala 8.21 118.4 - - 4.07 1.43 - - - - 181.8 55.1 18.3 - - - -104Glu 7.90 120.3 - - 4.02 2.79

2.592.612.21

- - - 178.0 59.7 29.0 38.1 - - -

105Leu 8.59 119.5 - - 4.11 1.871.57

1.57 0.860.84

- - 178.6 58.6 42.2 27.0 25.924.4

- -

106Arg 8.69 116.6 - 84.7 3.83 1.97 1.65 3.233.18

7.26 - 178.9 60.0 30.7 27.7 43.4 - -

107His 7.99 119.5 - - 4.20 3.303.27

- 6.76 - - 178.0 60.1 31.4 - 119.4 - -

108Val 8.77 119.4 - - 3.57 2.05 0.361.19

- - - 178.4 66.8 32.0 23.421.8

- - -

109Met 8.50 115.7 - - 4.24 2.392.08

2.942.46

- 1.92 - 178.8 58.4 30.7 34.2 - 18.4 -

110Thr 7.87 115.0 - - 3.99 4.29 1.22 - - - 178.0 66.8 68.4 21.3 - - -111Asn 7.80 122.2 111.4 - 4.44 2.81

2.78- 7.41

6.54- - 176.4 56.0 38.4 - - - -

112Leu 7.91 118.7 - - 4.41 1.961.85

1.86 0.810.80

- - 176.7 54.8 41.3 26.8 25.622.4

- -

113Gly 7.65 105.9 - - 4.183.75

- - - - - 174.2 45.9 - - - - -

114Glu 8.09 120.4 - - 4.45 1.951.66

2.222.02

- - - 174.9 54.2 30.1 35.3 - - -

115Lys 8.54 124.2 - - 4.34 1.75 1.31 1.66 2.97 - 175.5 55.8 31.8 24.4 29.0 41.9 -

116Leu 8.13 125.7 - - 4.82 1.581.52

1.6 0.810.80

- - 178.0 53.8 44.9 27.4 26.923.9

- -

117Thr 9.30 114.7 - - 4.46 4.71 1.32 - - - 175.5 60.6 70.8 21.8 - - -118Asp 8.87 120.8 - - 4.24 2.78

2.62- - - - 178.7 58.1 39.6 - - - -

119Asp 8.31 118.0 - - 4.42 2.662.55

- - - - 178.7 57.1 40.3 - - - -

120Glu 7.71 120.9 - - 4.00 2.421.91

2.392.25

- - - 179.9 59.3 30.4 37.9 - - -

121Val 8.09 120.8 - - 3.60 2.24 1.040.97

- - - 177.2 66.9 31.3 23.922.2

- - -

122Asp 7.99 119.4 - - 4.33 2.812.64

- - - - 179.1 57.6 40.5 - - - -

123Glu 7.96 119.5 - - 4.00 2.00 2.30 - - - 178.0 59.2 29.5 36.0 - - -

124Met 7.80 119.6 - - 4.01 2.291.97

2.752.37

- 1.98 - 178.8 59.4 33.2 32.2 - 17.1 -

125Ile 7.93 117.5 - - 3.49 2.06 1.591.140.66

0.69 - - 177.2 64.4 36.6 28.416.2

11.1 - -

126Arg 8.24 118.0 - 84.4 4.00 1.91 1.771.65

3.21 7.52 - 179.3 59.7 30.2 27.9 43.3 - -

127Glu 7.89 115.9 - - 3.99 2.06 2.442.27

- - - 177.2 58.6 29.8 36.6 - - -

128Ala 7.30 118.8 - - 4.45 1.42 - - - - 177.9 51.9 21.5 - - - -129Asp 8.00 117.8 - - 4.46 2.83

2.54- - - - 176.0 54.3 40.4 - - - -

130Ile 8.41 127.7 - - 3.98 2.03 1.631.380.92

0.85 - - 177.7 62.8 38.3 27.617.2

11.7 - -

131Asp 8.22 117.0 - - 4.54 3.062.62

- - - - 178.3 53.7 39.8 - - - -

132Gly 7.58 108.6 - - 4.003.83

- - - - - 175.3 47.4 - - - - -

133Asp 8.37 121.0 - - 4.61 3.112.52

- - - - 179.1 53.6 40.4 - - - -

134Gly 10.47 115.3 - - 4.003.40

- - - - - 172.2 46.1 - - - - -

135His 8.30 116.1 - - 5.05 2.872.69

- 6.33 - - 174.7 54.1 34.8 - - - -

136Ile 9.29 124.5 - - 5.17 2.08 1.551.260.94

0.75 - - 176.0 59.9 39.4 26.918.2

12.9 - -

137Asn 9.48 129.3 107.6 - 5.44 3.403.21

- 7.246.69

- - 175.4 51.1 37.9 - - - -

138Tyr 8.67 119.2 - - 3.51 2.402.01

- 6.45 6.53 - 176.1 62.8 37.5 - 132.3 118.1 -

139Glu 8.08 118.7 - - 3.66 2.091.99

2.312.30

- - - 180.4 60.1 29.1 36.7 - - -

140Glu 8.81 119.6 - - 3.99 2.592.11

2.842.45

- - - 179.3 58.3 29.9 36.9 - - -

141Phe 8.94 124.3 - - 3.97 3.503.23

- 6.91 7.04 6.64 176.5 61.5 39.9 - 131.7 131.3 131.6

142Val 8.52 119.4 - - 3.10 1.82 0.740.52

- - - 177.7 67.3 31.6 22.921.2

- - -

143Arg 7.32 116.2 - 84.5 3.91 1.85 1.57 3.21 7.39 - 178.6 59.0 29.9 27.4 43.2 - -

144Met 7.56 117.5 - - 4.11 2.092.04

2.432.42

- 1.79 - 177.3 57.8 33.1 31.3 - 17.1 -

145Met 7.68 116.1 - - 4.26 1.711.47

- - 1.71 - 177.8 55.5 31.9 - - 16.4 -

146Val 7.77 114.6 - - 4.26 2.21 0.900.89

- - - 176.3 61.8 32.3 20.919.9

- - -

147Ser 7.73 119.0 - - 4.39 3.893.89

- - - - 173.4 58.9 63.6 - - - -

148Lys 7.92 128.2 - - 4.19 1.831.70

1.39 1.66 2.97 - 181.1 57.4 33.8 24.5 28.9 42.0 -

Hovey et al, Calcium Triggers Reversal of Calmodulin on Nested Anti-Parallel Sites in the

IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2

Supplementary Table 3

Supports Table 2 - Structural Statistics for 2M5E.pdb

Chemical shifts of unlabeled Nav1.2IQp bound to (Ca2+)2-PCaM76-148. Red boxes indicate missing assignments.

Blue boxes indicate missing methylene or methyl proton assignments that may be degenerate.

Residue HN Hα Hβ Hγ Hδ Hε

1901Lys - - - - - 1902Arg - - - - - -1903Lys - - - - - -1904Gln 8.68 4.23 1.84

1.74- - -

1905Glu 8.57 4.18 2.062.01

2.35 - -

1906Glu 8.62 4.21 2.051.93

2.24 - -

1907Val 8.11 4.15 2.31 1.00 - -

1908Ser 8.78 4.08 3.883.79

- - -

1909Ala 8.47 3.98 1.45 - - -1910Ile 7.29 3.75 2.03 2.16

1.300.89

0.99 -

1911Val 7.47 3.50 2.12 0.980.89

- -

1912Ile 7.91 3.70 1.75 1.560.990.85

0.55 -

1913Gln 8.02 3.99 2.162.13

2.582.41

- 7.356.88

1914Arg 8.25 4.06 2.011.91

- 3.20 -

1915Ala 8.14 3.70 1.71 - - -1916Tyr 8.02 4.00 3.38

3.13- 6.90 6.77

1917Arg 7.87 4.05 2.161.96

1.921.60

3.323.26

-

1918Arg 8.39 3.86 2.021.91

1.70 3.223.17

-

1919Tyr 8.88 4.38 3.273.18

- 7.03 6.79

1920Leu 8.14 3.70 1.431.36

- 0.840.81

-

1921Leu 7.68 4.06 1.78 - - -

1922Lys 7.76 4.04 2.07 0.92 - -

1923Gln 8.18 4.30 1.831.73

1.43 - -

1924Lys 7.94 4.12 1.791.68

1.38 - -

1925Val - - - - - -1926Lys - - - - - -1927Lys - - - - - -

A,B Comparison of Metal-Chelating Residues in Site III of Apo/Mg2+-bound CaMC and Ca2+–bound CaMC CaM C-domain residues 76-148 (CaMC) in 8 structures were aligned using CaM residues 95-100 in Site III (93-104). CaMC is gray, Ca2+ ions are yellow spheres unless otherwise noted, and Mg2+ ions are gray. Residues are labeled according to the sequence of mammalian CaM.* For each labeled residue, the average distance (Å) between the metal ion (Ca2+ or Mg2+) and the coordinating oxygen is given. For clarity, only site III is labeled, but the geometry of ion-chelation in site IV is equivalent. A.  Structures of Semi-Open CaMC - apo (calcium-depleted) or ion-bound CaMC (Mg2+)4-CaM bound to the IQ motif of

NaV1.2(4DCK) and NaV1.5 (4OVN), and the IQ motif of Myosin VI (3GN4 chain H), and (Ca2+)4-CaM bound to the IQ motif of NaV1.2 (4JPZ) and NaV1.5 (4JQ0). Mg2+ ions are gray spheres and Ca2+ in 4JPZ and 4JQ0 are magenta spheres. Sidechains of metal-chelating residues in site III are highlighted with sticks: 4DCK - blue, 4OVN – cyan, 4JPZ - magenta, 4JQ0 - pink, and 3GN4 chain H - light blue.

B.  Structures of Open CaMC (Ca2+)4-CaM alone (1CLL), and (Ca2+)4-CaM bound to a CaMKII peptide (1CDM) or a Myosin VI “Insert 2” (3GN4 Chain B) Sidechains of Ca2+-chelating residues in site III are highlighted with sticks: 1CLL – orange, 1CDM – red, and 3GN4 chain B – salmon. Figures made with PyMOL.

C.  Calcium Fo-Fc Electron Density Maps The Fo-Fc density maps for two Ca2+ ions (yellow) located in sites III and IV in each calmodulin chain were contoured at +3 (green mesh, insufficient) and -3 (red mesh, excess) sigma to a radius of 5 Å from the center of each ion in calmodulin alone (1CLL), or in calmodulin bound to the IQ motif of NaV1.2 in 4JPZ [Chain C, Chain I]. Figures made with PyMOL. PDB files and electron density maps were obtained from PDB_REDO (http://www.cmbi.ru.nl/pdb_redo/) CaM residues 76-148 (gray) were aligned based on residues 117-128 (i.e., helix “G”).

* Mammalian CaM has 148 amino acids beginning with A1, and ending with K148. The Met listed as residue 1 in the UNIPROT

database is not found in mammalian CaM, or in the bacterially expressed sequence of mammalian CaM.

Hovey et al, Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2

Supplemental Figure S1 - Supports Figure 1

Hovey et al, Supplemental Figure S1

D95$2.6$Å$

D93$$2.4$Å$

N97$2.4$Å$ Y99$$

2.3$Å$

$E104$2.4$Å$

$E104$2.6$Å$

III IV

Helix$F$

Helix$E$Helix$E$

Helix$F$

D95$$2.3$Å$

N97$$2.5$Å$

D93$$2.3$Å$

Y99$2.3$Å$

E104$8.6$Å$

E104$8.9$Å$

III IV

+$Ca2+$

A.$$Apo/Mg2+$ B.$$Ca2+$

III IV III IV III IV

Helix G

Electron Density Maps: Red = Excess, Green = Insufficient 4JPZ: Calmodulin Chain C 1CLL: Calmodulin 4JPZ: Calmodulin Chain I

Helix G Helix G

11 10 9 8 7

130

125

120

115

110

105

1H (ppm)

15N (ppm

)

G25G61

E6/E45

K13F68 L4

L39

L18

T5/T44D22

G33G40

G59G23 T62

Hovey et al, Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2

Supplemental Figure S2 - Supports Figure 4

HSQC spectra of 13C,15N-(Ca2+)4-CaM(1-148)-Nav1.2IQp vs. 13C,15N-(Ca2+)2-CaM(1-75) HSQC overlay of full-length CaM saturated with calcium and Nav1.2IQ [(Ca2+)4CaM-Nav1.2IQp; black] with free CaMN [(Ca2+)2-CaM(1-75); blue]. Peaks that are nearly identical are circled in green. The high level of agreement between these two spectra indicates that few residues in the N-domain of CaM are perturbed by CaM binding to the IQ motif and the C-domain makes most contacts in the CaM-Nav1.2IQp interface.

C

N

N

Hovey et al, Supplemental Figure S2

final_avmin_01_ramachand.ps ** Selected residues only

PROCHECK-NMR

B

A

L

b

a

l

p

~p

~b

~a

~l

b

~b

b~b

~b

-180 -135 -90 -45 0 45 90 135 180

-135

-90

-45

0

45

90

135

180

Ramachandran Plotfinal_avmin (1 models)**

Phi (degrees)

Psi (

degr

ees)

Plot statisticsResidues in most favoured regions [A,B,L] 77 90.6%Residues in additional allowed regions [a,b,l,p] 8 9.4%Residues in generously allowed regions [~a,~b,~l,~p] 0 0.0%Residues in disallowed regions 0 0.0% ---- ------Number of non-glycine and non-proline residues 85 100.0%Number of end-residues (excl. Gly and Pro) 0 Number of glycine residues (shown as triangles) 5 Number of proline residues 0 ---- Total number of residues 90

Based on an analysis of 118 structures of resolution of at least 2.0 Angstromsand R-factor no greater than 20%, a good quality model would be expected

to have over 90% in the most favoured regions.

Hovey et al, Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2

Supplemental Figure S3 - Supports Figure 5

Procheck analysis of the minimized average structure in 2M5E

Hovey et al, Supplemental Figure S3

Hovey et al, Calcium-Triggered Reversal of Calmodulin on Nested Anti-Parallel Sites in the IQ Motif of the Neuronal Voltage-Dependent Sodium Channel NaV1.2

Supplemental Figure S4 - Supports Figure 6 NOESY Overlay of Nav1.2IQp Bound to CaM ± Ca2+ Overlays comparing two regions of the doubly 12C,14N filtered NOESY spectra of Nav1.2IQp bound to apo (green) and Ca2+ saturated (black) CaM. Crosspeaks are shown (a) between upfield and downfield aliphatic protons and (b) between amide plus aromatic protons and aliphatic protons. The significant differences suggest that the peptide is in a very different chemical environment when bound to CaM in the absence and presence of calcium.

a

b

Hovey et al, Supplemental Figure S4